Chemosphere 144 (2016) 635–644

Contents lists available at ScienceDirect

Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Biosurfactant-enhanced bioremediation of aged polycyclic aromatic

hydrocarbons (PAHs) in creosote contaminated soil
Fisseha Andualem Bezza, Evans M. Nkhalambayausi Chirwa∗
Water Utilisation and Environmental Engineering Division, Department of Chemical Engineering, University of Pretoria, Pretoria 0002, South Africa

h i g h l i g h t s g r a p h i c a l a b s t r a c t

• A crude biosurfactant was exoge-

nously applied to enhance biodegra-
dation of PAHs.
• Biosurfactants produced in situ under
growth limitation encouraged longer
stability.
• Growth limited in situ production
was effective in preventing microbial
scavenging.
• High removal rate of high-molecular
weight PAHs was observed in the
bioreactors.

a r t i c l e i n f o a b s t r a c t

Article history: The potential for biological treatment of an environment contaminated by complex petrochemical con-
Received 26 November 2014 taminants was evaluated using creosote contaminated soil in ex situ bio-slurry reactors. The efficacy of
Received in revised form 28 July 2015
biosurfactant application and stimulation of in situ biosurfactant production was investigated. The bio-
Accepted 5 August 2015
surfactant produced was purified and characterised using Fourier transform infrared (FTIR) spectroscopy.
Available online 25 September 2015
Biosurfactant enhanced degradation of PAHs was 86.5% (with addition of biosurfactant) and 57% in con-
Handling editor: Chang-Ping Yu trols with no biosurfactant and nutrient amendments after incubation for 45 days. A slight decrease in
degradation rate observed in the simultaneous biosurfactant and nutrient, NH4 NO3 and KH2 PO4 , supple-
Keywords:
Biosurfactant mented microcosm can be attributed to preferential microbial consumption of the biosurfactant supple-
Creosote degradation mented. The overall removal of PAHs was determined to be mass transport limited since the dissolution
In situ biosurfactant production rate caused by the biosurfactant enhanced the bioavailability of the PAHs to the microorganisms. The con-
Bio-slurry reactor sortium culture was predominated by the aromatic ring-cleaving species Bacillus stratosphericus, Bacillus
subtilis, Bacillus megaterium, and Pseudomonas aeruginosa.
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction (Li et al., 2014). Soils from many sites, such as areas of coal stor-
age, coke oven plants, manufactured gas plants and areas of coal
Polycyclic aromatic hydrocarbons (PAHs) are of human health tar spillage are of high contamination of PAHs (Li et al., 2010).
concern due to their known or suspected genotoxic, mutagenic A typical source of PAH contamination in soil is coal-tar creosote
or carcinogenic effects (Hu et al., 2012). PAHs are mainly derived which was commonly used to preserve and waterproof railway ties
from anthropogenic activities, such as biomass burning, incomplete and power line poles. Creosote is a complex mixture of over 200
fossil fuel combustion, oil spills and some industrial processes compounds, predominantly PAHs, as well as phenolic and aromatic
nitrogen and sulphur compounds. PAHs comprise 85% of the cre-
∗
osote composition by weight and up to 30 different PAH species
Corresponding author.
E-mail address: Evans.Chirwa@up.ac.za (E.M. Nkhalambayausi Chirwa).
can be released from one creosote source (Melber et al., 2004).

http://dx.doi.org/10.1016/j.chemosphere.2015.08.027
0045-6535/© 2015 Elsevier Ltd. All rights reserved.
636 F.A. Bezza, E.M. Nkhalambayausi Chirwa / Chemosphere 144 (2016) 635–644

Several treatment strategies involving biological, physicochemical, 15 Orbital Shaker (Labcon Laboratory Services, South Africa). The
and thermal processes have been developed to remediate con- cultures were grown at a constant temperature of 30 ± 1 °C and
taminated sites. Methods such as incineration, excavation, land- pH 7. Five successive subcultures each grown for 7 days were per-
filling and storage are expensive, inefficient, and often exchange formed by transferring 10 mL of enriched culture into 100 mL fresh
one problem for another (Bustamante et al., 2012). Biological treat- media and 5% (v/v) fresh creosote.
ment, on the other hand, offers low cost and more environmentally
friendly alternative since the organic components that are respon- 2.2. Growth media
sible for the toxicity may be completely mineralized to CO2 and
H2 O through known biological degradation pathways (Chirwa and The mineral salt medium (MSM) was prepared by adding (in
Wang, 2000). 1 L distilled water: 6.0 g (NH4 )2 SO4 ; 0.4 g MgSO4 ·7H2 O; 0.4 g
However, biological remediation can be limited by the bioavail- CaCl2 2H2 O; 7.59 g Na2 HPO4 2H2 O; 4.43 g KH2 PO4 ; and 2 mL of
ability of soil-bound PAHs due to their low aqueous solubility, high trace element solution (Trummler et al., 2003). The trace element
hydrophobicity and strong sorption to soil, which is exacerbated solution consisted of (in 1 L distilled water: 20.1 g EDTA (disodium
by the long ageing of contaminants in field-contaminated soils salt); 16 g FeCl3 ·6H2 O; 0.18 g CoCl2 6H2 O; 0.18 g ZnSO4 7H2 O;
(Zhu and Aitken, 2010). Surface-active compounds may be used 0.16 g CuSO4 5H2 O, and 0.10 g MnSO4 H2 O. In order to create solid
to increase the bioavailability of otherwise poorly accessible car- media for plating and streaking, 23 g Bacto Agar (BA) was added to
bon sources, thus helping to overcome the diffusion-related mass 1 L MSM and the mixture was sterilized by autoclaving at 121 °C
transfer limitations (Singh et al., 2007; Szulc et al., 2014). Produc- for 15 min. Agar was poured into the plates at 40–45 °C.
tion of biosurfactants by bacteria is considered an important mi-
crobial strategy that influences the bioavailability of hydrophobic 2.3. Culture isolation and purification
chemicals by changing the surface properties of bacterial cell or
by dissolving and emulsifying these hydrophobic hydrocarbons, as Single colonies from serially diluted samples from the enrich-
well as releasing the trapped hydrocarbons of porous medium in ment cultures were tested for biosurfactant production using Drop-
contaminated soil (Xia et al., 2014). Surfactants are amphiphilic Collapse method (Chandankere et al., 2014). The ‘drop collapse’ as-
molecules consisting of hydrophobic and hydrophilic moieties that say relies on the destabilization of liquid oil droplets by surfac-
tend to interact with interfaces of various polarities and reduce the tants. An oil droplet will be repelled by a hydrophilic surface due
surface and interfacial tension and increase the solubility, mobil- to surface tension at the water oil interface. If the liquid on the
ity, bioavailability and subsequent biodegradation of hydrophobic surface contains surfactants, the surfactant will weaken the surface
or insoluble organic compounds (Singh et al., 2007; Hazra et al., tension resulting in the tendency for the droplet to spread or col-
2012; Xia et al., 2014). In recent years, interest in biosurfactants lapse.
has increased due to their several advantages over their chemi- Each fully developed colony was picked by heat sterilized wire
cal counterparts, such as biodegradability and low toxicity, higher loop and transferred into 10 mL sterile medium supplied with
foaming; specific activity at extreme temperatures, pH, and salinity 5% (v/v) glycerol to grow for 3 days. Four microlitres of crude
and ability to be synthesized from renewable feed-stocks (Shavandi oil was applied to the well regions delimited on the covers of
et al., 2011). 96-well micro-plate lids and allowed to equilibrate for 24 h. Five
The soil environment represents the most difficult medium to microlitres of the cell free culture broth was transferred to the
remediate. The objective of the current study was to test the effect oil coated regions and the drop size was observed 2 min later
of biosurfactant on the bioavailability and subsequent biodegrad- (Bodour and Miller-Maier, 1998). If the cell free broth results in
ability of toxic and hazardous PAH components of Creosote in a soil droplet collapse within 2 min the result was considered positive
environment. The ability of biosurfactant to enhance degradation for biosurfactant production and were sent for characterization us-
of PAHs in creosote is investigated using cultures enriched on PAH ing 16S rRNA gene sequencing. All genetic analysis was conducted
containing medium. The cultures’ performance in degrading PAHs in the Microbiology Laboratory at the Department of Microbiology
from creosote is evaluated using bio-slurry reactors. Biosurfactant and Plant Pathology (University of Pretoria). Partially sequenced
producing bacteria in the consortium were identified as close ho- amplified 16S rDNA fragments were compared with other gene
mologs of Bacillus subtillis, Bacillus stratosphaericus, Bacillus mega- sequences in GenBank using (http://www.ncbi.nlm.nih.gov/BLAST/)
terium and Pseudomonas aeruginosa using 16S rRNA gene sequenc- and aligned with gene sequence of our isolates. The aligned se-
ing. Once it is established that the above microbial cultures indeed quences were used to construct a distance matrix, after the gen-
achieves the degradation of the PAHs with the aid of biosurfactant, eration of 1000 bootstrap sets that was subsequently used to
a delivery system for the microorganism into real environment will construct a phylogenetic tree using the neighbour-joining method
be developed. MEGA version 6 software (Tamura et al., 2013). Then complete 16S
rDNA sequences of the strains CB1, CB2, CB3, CB4, CN1, CN2, CN3
2. Material and methods and CN5 have been deposited in the GenBank database under the
accession numbers KP793922, KP793923, KP793924, KP793925,
2.1. Enrichment of microbial consortium KP793926, KP793927, KP7939228, KP793929 respectively.

Creosote degrading bacteria were cultured from soil obtained 2.4. Crude biosurfactant production
from wood treatment plant in Pretoria West (Pretoria, South
Africa). Due to the creosote contamination at the site, high levels P. aeruginosa CB1 strain was selected based on Drop Collapse
of PAHs, PCBs and other petrochemical organic contaminants were test as efficient biosurfactant producer and was grown in 1 L of
detected in the soil. The plant has been utilized for over 20 years (Phosphate -limited) MSM, 2% (w/v) of the inoculum and 60 g L−1
therefore organisms in the soil are expected to be acclimated to of glycerol as a carbon source. The culture was incubated for 6 days
PAHs. In order to selectively isolate efficient creosote degraders 5 g at 37 °C, pH 7, with shaking at 150 rpm in an Orbital Shaker (Lab-
contaminated soil from the polluted ground was inoculated into con Laboratory Services). The biosurfactant was extracted using the
100 mL of mineral salt medium (MSM) containing 5% (v/v) cre- method derived earlier by Zhang and Miller (1992). The culture at
osote as a source of carbon and energy. The culture was grown for day 6 was harvested by centrifuging at 12,000 rpm for 20 min at
7 days under continuous shaking at 120 rpm in a Labcon SPL-MP 4 °C. The cell free supernatant was acidified to pH 2.0 using 6 M
 F.A. Bezza, E.M. Nkhalambayausi Chirwa / Chemosphere 144 (2016) 635–644 637

Table 1
Biodegradation of PAHs in creosote contaminated soil after incubation of the soil for 25 days; the results represent the mean ± standard deviation of the three replicates.

Compound Initial conc. mg kg−1 Final concentration in the different bioslurry reactors (mg kg−1 )

R1a Removal % R2 Removal % R3 Removal % R4 Removal %

Nap 425 ± 16 112 ± 10.6 74 ± 0.7 210 ± 15 51 ± 0.3 162 ± 5.6 62 ± 0.2 296 ± 21 30 ± 0.2
Ace 272 ± 11 41 ± 3.9 85 ± 0.9 46 ± 2.8 83 ± 0.4 60 ± 12 78 ± 3.2 163 ± 17 40 ± 0.5
Flou 383 ± 25 55 ± 5.7 86 ± 1.2 107 ± 15 72 ± 1.7 116 ± 19 70 ± 2.2 211 ± 31 45 ± 1.2
Phe 233 ± 3.6 27 ± 0.56 88 ± 0.1 44 ± 3.3 81 ± 0.5 44 ± 07 81 ± 2.1 95 ± 9.4 59 ± 0.6
Ant 131 ± 7.8 42 ± 7.2 68 ± 2.2 49 ± 7.2 63 ± 1.6 61.5 ± 4.2 53 ± 0.4 81 ± 7.7 38 ± 0.5

Flr 321 ± 19 58 ± 8.4 82 ± 2.0 141 ± 10 56 ± 0.5 160 ± 7.8 50 ± 0.3 305 ± 51 5 ± 0.2
Pyr 703 ± 35 127 ± 9.4 82 ± 0.7 202 ± 21 71 ± 0.9 197 ± 17 72 ± 0.7 303 ± 21 57 ± 0.4
BaA 145 ± 17 30 ± 5.3 79 ± 3.6 56 ± 13 61 ± 4.1 57 ± 7.8 61 ± 2.1 82 ± 09 43 ± 1.1
Chr 62 ± 7.8 12 ± 2.4 81 ± 3.4 17 ± 2.8 73 ± 3.1 16 ± 1.4 74 ± 1.7 39 ± 5.6 37 ± 1.3

BbF 57 ± 7.4 39 ± 4.2 32 ± 0.9 43 ± 06 25 ± 0.9 45 ± 05 21 ± 0.5 37 ± 8.3 35 ± 2.4

BkF 152 ± 11 38 ± 7.6 75 ± 3.4 45 ± 5.4 70 ± 1.4 59 ± 08 61 ± 1.4 61 ± 11 60 ± 2.3
BaP 24 ± 3.2 18 ± 3.2 25 ± 1.2 20 ± 3.5 17 ± 0.8 22 ± 4.6 8 ± 0.4 23 ± 4.2 04 ± 0.2
DahA 56 ± 07 20 ± 4.9 64 ± 4.8 17 ± 3.7 70 ± 4.4 24 ± 03 57 ± 1.8 27 ± 7 52 ± 4.3
BPer 97 ± 11 15 ± 3.1 85 ± 4.7 16 ± 2.6 84 ± 3.3 31 ± 5.4 68 ± 3.1 66 ± 6 32 ± 0.7
IcdP 3.6 ± 0.5 –b – – – – – – –

Total (mg kg−1 ) 3064.6 ± 14 635 ± 5.9 79 ± 2.5 1013 ± 9.4 67 ± 2.1 1055 ± 8.8 66 ± 1.7 1789 ± 18.8 42 ± 1.5

Naph, Naphthalene; Phe, Phenanthrene; Pyr, Pyrene; BbF, Benzo[b]fluoranthene; DahA, Dibenz[a,h]anthracene; Ace, Acenaphthene; Ant, Anthracene; BaA, Benz[a]anthracene;
BkF, Benzo[k]fluoranthene; BPer, Benzo[ghi]perylene; Flou, Fluorene; Flr, Fluoranthene; Chr, Chrysene; BaP, Benzo[a]pyrene; IcdP, indeno(1,2,3-cd)pyrene.
a
Reactor labels, R1 = reactor charged with biosurfactant and cells added on day 2, R2 = reactor charged with biosurfactants and nutrients NH4 NO3 and KH2 PO4 at time
zero and cells on day 2, R3 = reactor charged with nutrients only and cells added on day 2, and R4 = Control reactor with cells added on day 2 (biotic control).
b
‘––’ concentration was below instrument detection limit.

HCl and allow precipitate to form at 4 °C overnight. The biosur- IL, USA). The amount of protein is measured by reading the ab-
factant was then extracted using equal volume of 2:1 (v/v), mix- sorbance at 595 nm in a microplate reader (Multiskan Ascent V1.24
ture of Chloroform–Methanol solution. The organic phase was then plate reader) based on a standard curve using bovine serum al-
transferred to a round-bottom flask connected to a rotary evapora- bumin as a standard (Meng et al., 2012). Protein content was ex-
tor to remove the solvent which yielded a viscous brown-coloured pressed as micrograms of protein per millilitre of sample. All the
biosurfactant product. About 9.80 g of crude biosurfactant was ex- experiments were conducted in triplicates.
tracted per litre of culture medium using this method.
2.6. Evaluation of organics in bio-slurry
2.5. Evaluation of molecular structure of biosurfactant
The soil sample was collected from the top surface layer
2.5.1. Fourier transform infrared (FTIR) spectroscopy (15 cm) at a wood impregnation plant in the outskirts of Preto-
The chemical structure and components of the crude bio- ria (Gauteng, South Africa). A Total Organic Carbon (TOC) Analyser
surfactant sample were determined using Fourier transform in- (Model TOC-VWP, Shimadzu Corporation, Kyoto, Japan) was used
frared (FTIR) spectroscopy (Perkin Elmer 1600 FTIR) equipped with to determine organic content of a washed eluent whereas a gravi-
an Attenuated Total Reflectance (ATR) Crystal Accessory (Perkin metric method was used to determine total organic on the sam-
Elmer, Connecticut, USA). The IR scan was performed over 400– ple. The TOC analyzer was calibrated by dissolving different pro-
4000 cm−1 wave number range with a resolution of 2 cm−1 . The portions of a 1000 mg L−1 potassium hydrogen phthalate stock so-
reflectance spectra were recorded and averaged over 32 scans, us- lution in concentrations ranging from 0 to 5 mg L−1 in a 100 mL
ing the total internal reflectance configuration with a HarrickTM volumetric flask prior to analysing for total carbon. The approxi-
MVP-PRO cell consisting of a diamond crystal. Spectra were viewed mate organic composition in the soil was 210 g kg−1 TOC of which
in OMNIC software. 3.062 g kg−1 (1.5%) was PAHs. To determine the PAHs in the soil,
5 g samples were extracted using the USEPA Method 3550B which
2.5.2. Thin layer chromatography (TLC) was developed for extracting non-volatile and semi-volatile organic
The crude extracts were further purified through a Silica gel compounds from solids (U.S.EPA, 1996) as described in section 2.7
(60–200; Merck KGaA) column. Elution was carried out with chlo- and HPLC analysed. Most of the common PAHs were detected in
roform/methanol mixtures with step-wise increase of methanol the soil sample from the site at levels shown in Table 1. Notably,
from 75:25 to 50:50 (v/v) solvent (200 mL each) at a flow rate naphthalene (Nap), acenaphthene (Ace), fluorene (Flu), phenan-
of 1 mL/min. Column purified biosurfactant was dissolved in threne (Phe), anthracene (Ant), fluoranthene (Flr) and pyrene (Pyr)
methanol and 10 μl of this solution was subjected to TLC analy- were among the most abundant PAHs detected in the soil. Perkin
sis on silica gel 60 plates (Merck) with chloroform–methanol–H2 O Elmer 2400 CHN/O Elemental Analyser was used to determine the
(65:25:4, v/v/v) as the mobile phase. For the detection of peptides, soil samples percentage composition of carbon and nitrogen, which
the dry plates were sprayed with a solution of 0.25% ninhydrin in showed that the amount of nitrogen in the soil sample was lower
acetone and kept at 115 °C for 5 min (Xia et al., 2014). than the detection limit of the instrument.

2.5.3. Protein assay 2.7. Bio-slurry reactor operation

After column purification of the crude extracts through a Sil-
ica gel (60–200; Merck KGaA) Protein assay was conducted to con- Degradation studies were conducted in five bio-slurry reactors
firm lipopeptidal nature of the biosurfactant and determine con- using 400 g of soil suspended in 1000 mL distilled water. 2 L Erlen-
centration of protein in the sample. Concentration of protein was meyer flasks were used as bioreactors with continuous mixing us-
measured by Coomassie (Bradford) Protein Assay kit (Rockford, ing overhead mechanical mixers. Reactor 1 was supplemented with
638 F.A. Bezza, E.M. Nkhalambayausi Chirwa / Chemosphere 144 (2016) 635–644

3 g kg−1 crude biosurfactant (no added nutrients), Reactor 2 was of n-hexane, the bottle was vortex-mixed for 5 min followed by
supplemented with 3 g kg−1 crude biosurfactant and biostimulated centrifugation at 10,000 rpm for 10 min to separate the aqueous
with 11.5 g NH4 NO3 and 1.5 g KH2 PO4, Reactor 3 was biostimulated and organic phase. After centrifugation, the supernatant was de-
with 11.5 g NH4 NO3 and 1.5 g KH2 PO4 (no biosurfactant), Reactor canted into a separatory funnel. The lower aqueous phase was dis-
4 was the un-amended biotic control, and Reactor 5 was the ster- carded and the combined organic extract obtained from repeated
ilized abiotic control. Reactor 2 and 3 were supplemented with the extractions was passed through anhydrous sodium sulphate. Sub-
nutrients twice at the beginning of the experiment and at the third sequently, n-hexane was evaporated to dryness under a nitrogen
week to obtain a C:N:P ratio of 100:10:1 in the soil (Cookson and stream and the Phenanthrene extracted was dissolved in an equiv-
John, 1995). All reactors were installed in a water bath at constant alent volume of HPLC grade acetonitrile. The extract in acetoni-
temperature of 37 ± 1 °C. The sterile abiotic control (Reactor 5) trile (HPLC mobile phase) was injected into an HPLC through a
was prepared by autoclaving the soil slurry at 121 °C (2.0 bar) for 0.22 μm polytetrafluoroethylene (PTFE) filter syringe. The concen-
15 min. 10 mL aliquots of the PAH degraders from late-log precul- tration of the PAH was calculated from 4-point standard calibration
tures was inoculated into the four bio-slurry reactors apart from curves.
Reactor 5 on day 2 of incubation to achieve a final cell density
of (10 7 CFU/mL). All reactors were vigorously mixed using over- 2.8.2. Liquid phase biodegradation assay
head mechanical mixers. Water lost in the reactors via evapora- The experiment was conducted to evaluate the impact of the
tion was replaced daily to keep the working volume constant. The biosurfactant on the biodegradation of Model PAH. 5 ml of the
make-up was not used to adjust for volume loss due to drawing of microbial consortium was used to inoculate 100 mL of mineral
samples. salt medium (Trummler et al., 2003), supplemented with Pyrene
Samples (25-mL aliquots) of the bio-slurry contents were drawn to achieve a final concentration of 100 mg L−1 . The cultures were
at predetermined intervals and centrifuged at 6000 rpm for incubated at 37 °C and shaker speed of 120 rpm for 16 days. Pe-
10 min. The harvested soil was air-dried after which 5 g was sub- riodically the residual PAH was sacrificially extracted using the
jected to ultrasonic extraction followed by High Performance Liq- method described by Ghosh et al. (2014). Briefly, the entire content
uid Chromatograph (HPLC) analysis of PAHs in the extracts. The of a set of flasks in duplicate were extracted twice with n-hexane
samples were extracted using the USEPA Method 3550B which (extraction ratio 1:1). Immediately after addition of n-hexane, the
was developed for extracting non-volatile and semi-volatile organic bottle was vortex-mixed for 5 min followed by centrifugation at
compounds from solids (U.S.EPA, 1996). The method involved air 10,000 rpm for 10 min to separate the aqueous and organic phase.
drying and homogenizing a 5 g soil sample and mixing with 30 mL After centrifugation, the supernatant was decanted into a sepa-
of a solvent hexane: acetone (1:1 v/v) in flask, followed by sonica- ratory funnel. The lower aqueous phase was discarded and the
tion at 50–60 Hz at 55 °C for 60 min (M1800 Ultrasonic bath, USA). combined organic extract obtained from repeated extractions was
The sample was transferred to centrifuge tubes and the soil parti- passed through anhydrous sodium sulphate, and the residuals were
cles were removed from the liquid by centrifugation at 2000 rpm HPLC analyzed as described above.
for 10 min. The organic layer containing the extracted compounds
was drawn off with a pipette. The extraction was performed twice
2.9. Analytical methods
before disposing the solids to achieve thoroughness of removal of
PAHs from the soil.
2.9.1. HPLC analysis
The final extract from each sample was vacuum-filtered to re-
PAHs in the aquatic phase were analysed using the Waters 2695
move particles that might have been integrated into the super-
HPLC equipped with a Photo Diode Array (PDA) detector (Waters
natant during centrifugation. The cleaned extract was evaporated
Corporation, Massachusetts, USA). Separation of compounds was
to dryness under a nitrogen stream and re-dissolved in 5 mL of
performed on a Waters PAH C18 column (4.6 mm × 25 cm with
acetonitrile. The extract in acetonitrile (HPLC mobile phase) was
5 μm packing) (Waters Corporation) at a column temperature of
injected into an HPLC through a 0.22 μm polytetrafluoroethylene
25 °C and 254 nm wavelength. The chromatographic conditions
(PTFE) filter syringe. The concentration of each PAH was calculated
applied were: 0–1 min, 70% acetonitrile, A:30% ultrapure water,UP,
from 4-point standard calibration curves.
isocratic; 1–25 min, 70% A:30% UP – 100% A, linear gradient; 25–
35 min, 100% A isocratic; 35–40 min, 100% A – 70% A:30% UP,
2.8. Biosurfactant enhanced desorption and biodegradation kinetics
linear gradient; and finally; 40–45 min, 70% A:30% UP isocratic
assays
back to the initial condition and recondition the column at a flow
rate of 1.0 mL min−1 . The detection limit of the HPLC system was
2.8.1. Mass transport kinetics
0.01 mg L−1 . All tests were conducted in triplicate with uninoc-
Batch experiments were conducted in duplicate to determine
ulated controls to monitor the abiotic/volatilization photodegrada-
the desorption and subsequent degradation of model PAH, phenan-
tion losses and total recovery of contaminants.
threne (PHE), using 500 mL Erlenmeyer flasks. A mass of 150 g of
the soil sample from the wood treatment plant was weighed into
each flask containing 300 mL of mineral salt medium (Trummler 2.9.2. Emulsification index (E24 )
et al., 2003), 50% (w/v) with different amount of Lipopeptide Emulsification was measured by the method reported earlier by
(700 mg L−1 ≈ 4.66 g kg−1 of soil and 400 mg L−1 ≈ 2.67 g kg−1 Cooper and Goldenberg (1987). Briefly, equal volumes of reactor
of soil). The flasks were shaken on a rotary shaker at 120 rpm un- slurry and hydrocarbons (kerosene, diesel or hexane) were mixed
der darkness at 30 °C. Samples (25-mL aliquots) of the bio-slurry and vortexed at high speed for 5 min followed by incubation at
contents were drawn at predetermined intervals and centrifuged 25 °C for 24 h. The emulsification index value (E24 ) is given as per-
at 6000 rpm for 10 min. The PAH in the solid phase was extracted centage of height of emulsified layer (mm) divided by total height
following the USEPA Method 3550B as described in the Material of the liquid column.
and Methods section (2.7).
The PAH fraction in the liquid phase was extracted using the 2.9.3. Viable biomass determination
method described by Ghosh et al. (2014). Briefly, 30 mL super- Quantitative analysis of viable biomass was conducted gravi-
natant was decanted in to 150 mL bottle after centrifugation and metrically as a function of volatile suspended solids (VSS). The VSS
extracted twice with 30 mL n-hexane. Immediately after addition from soil samples was determined as the difference in dry weight
 F.A. Bezza, E.M. Nkhalambayausi Chirwa / Chemosphere 144 (2016) 635–644 639

reagent in Bradford assay and a concentration of 55 μg mL−1 of

protein was determined from the standard curve.

3.3. Bioavailability and biodegradability of PAHs

The concentration of 15 PAHs in creosote was monitored against

purchased standards using the HPLC. Chromatographic peaks in
the sample are matched with peaks and their retention times
of known standards of the PAHs as shown in the chromatogram
(Supplementary Figure S2). Short-to intermediate-term degradabil-
ity of PAHs in the solid phase of the soil slurry was determined
after 25 days of incubation (Table 1). The long-term performance
of the culture was evaluated after incubation for 45 days (Table 2).
The data in Tables 1 and 2 show that degradability of the PAHs
decreased with increasing ring-number. The decrease in degrada-
Fig. 1. Fourier transform infrared absorption spectra of crude biosurfactant pro-
tion of PAHs with increasing ring number was also confirmed ear-
duced by a pure colony of Pseudomonas aeruginosa CB1. lier by Tikilili and Chirwa (2011). At 25 days of incubation the
highest degradation efficiency was observed for Phenanthrene (88%
removal) in the biosurfactant only amended reactor (Reactor 1).
of samples of known volume after igniting thoroughly dried sam- The pattern of degradation of PAHs was studied by evaluating the
ples at 600 °C in a furnace. Results were calibrated against colony degradation rate of phenanthrene in the two phases (solid phase
forming units from a heterotrophic (pour) plate method on Luria– and aqueous phase) against time (Fig. 4 ). Phenanthrene (3-ring)
Bettani (LB) and Plate Count (PC) agar from soil samples dispersed was chosen as the model compound since it is in the intermedi-
in sterile saline (0.85% w/v NaCl) solution. The pour plate method ate range of rings and its degradation could show a realistic pat-
was conducted based on the method described in the Standard tern of the degradation of the PAHs. The presence of the biosurfac-
Methods for the Examination of Water and Wastewater (APHA, tant resulted in the increase of the aqueous phase concentration of
2005). the PAH, which resulted in the increased bioavailability and its fast
subsequent degradation.
Nevertheless ex situ supplementation of biosurfactant beyond
3. Results and discussion
optimum level could result in toxicity levels above the tolerance
of the organisms. The toxicity may be due to the biosurfactant
3.1. Biosurfactant activity and classification
overdose or increased stress caused by the solubilized PAHs. Shin
et al. (2006) reported that rhamnolipid inhibited degradation of
Before going into detail investigation of the biosurfactant and
phenanthrene by a two-species consortium of Sphingomonas and
culture performance, it was necessary to determine the nature of
Paenibacillus sp., even though in pure culture the rhamnolipid
the crude biosurfactant produced by the isolate. The FTIR scans
inhibited only Sphingomonas sp. The authors suggested that the
showed spectrum absorption (reverse peaks) at wave numbers
increased stress caused by the solubilized phenanthrene or the
of 1458, 3217, 1642 cm−1 indicating a strong signature for the
increased toxicity of rhamnolipid in the presence of solubilized
(–CH3 ,CH2 –), –NH–, and CO–N components, respectively (Fig. 1).
phenanthrene could have resulted in the inhibitory effect in the
Additional reverse peaks in the wave number range 1100–
case of Paenibacillus sp. Observation of toxicity of increased biosur-
1040 cm−1 indicated the presence of amine groups. The nature of
factant concentration on the bacteria was reported by (Vasileva-
compound that best fits this description is the lipopeptide. Further
Tonkova et al., 2011). The authors reported complete inhibition of
analysis is underway with a purified sample for the biosurfactant
the growth of B. subtilis 168 at higher concentrations of biosur-
characterization.
factant. Biosurfactant induced toxicity can be caused due to over-
dosage beyond threshold level which causes disruption of cellular
3.2. Microbial culture membranes by interaction with lipid component and reactions of
surfactant molecules with proteins that is essential to the func-
Based on the 16S rRNA Gene Sequencing, the strains isolated tioning of the cell (Hames et al., 2014). It is important therefore
from the Creosote contaminated soil contained efficient PAH de- for the dissolution process to be optimized and experimentally de-
grading and biosurfactant producing species like Bacilli – i.e., Bacil- termined to avoid toxicity and just fast enough to avoid starvation
lus stratosphericus, Bacillus subtilis, and B. megateriumi, Ochrobac- of cells since the PAHs in this system were the only carbon and
trum sp. and P. aeruginosa, (Fig. 2). These are only few members energy sources.
of the consortium which were identified as efficient biosurfactant Samples from the bioreactors were assayed for biosurfactant
producers. The Lipopeptidal structure of biosurfactant produced production utilizing Emulsification index (E24 ) measurement us-
by P. aeruginosa in this study showed an identical FTIR spectrum ing the method described by Cooper and Goldenberg (1987). The
as the one produced by B. subtillis (Joshi et al., 2008). Thavasi emulsification index of the sample was determined at day 18 and
et al. (2011) also arrived at a similar conclusion alluding to the was found out to be 90% and 96%, with Hexane in the biostimu-
lipopeptide structure of the biosurfactant produced by P. aerugi- lated and instantaneous biosurfactant and fertilizer stimulated re-
nosa species. actors respectively. This continuous in situ biosurfactant production
In the TLC analysis treatment with ninhydrin reagent showed is due to growth limitation following nitrogen depletion and reach-
positive reactions and revealed pink spot at Rf value of 0.74 ing stationary growth phase which is in line with previous ob-
(Supplementary Figure, Figure S1a). Signifying that the biosurfac- servations reported (Hwang and Cutright, 2002). However in the
tant consisted of peptide moieties and showing the lipopeptidal second round supplementation of the nutrients on day 25 the
nature of the biosurfactant. The results (Supplementary Figure, Fig- emulsification observed was dissipated. The observed dissipation
ure S1b) showed positive reaction of the sample with Bradford may be attributed to preferential microbial consumption of the
640 F.A. Bezza, E.M. Nkhalambayausi Chirwa / Chemosphere 144 (2016) 635–644

Fig. 2. Phylogenetic tree diagram showing the identity of biosurfactant producing and PAH degrading species identified as most closely associated with Pseudomonas aerugi-
nosa, Bacillus, Paenibacillus and Ochrobactrum sp.

biosurfactant produced indicating the necessity of nutrient limita- contaminated by the wood mill (the bio-slurry soil source) is lack-
tion for in situ biosurfactant production. ing the nutrients and the low bioavailability of the PAHs is respon-
Amendment of the bioslurry reactor with nutrients (11.5 sible for the observed poor performance even if it has been inocu-
NH4 NO3 and 1.5 KH2 PO4 ) and biosurfactant (Reactor 2) showed lated with the same consortium. In actual application, PAH degrad-
relative inhibition at the beginning as indicated by the decrease ing organisms for the clean-up of the site have to be sourced from
in the degradability of the compounds (51% naphthalene and 81% the sources which had been acclimated through long term expo-
phenanthrene at day 25) as opposed to removal in the reactor with sure. A creosote exposed microbial culture as applied in this study
biosurfactant amendment but without nutrient amendment (Reac- could serve the purpose. No removal of PAHs from the solid phase
tor 1:74% naphthalene and 88% phenanthrene removed). The other was observed in the sterilized abiotic control (Reactor 5) showing
3 and 4 ring PAHs removal was also comparatively inhibited in Re- that abiotic losses were insignificant.
actor 2 as opposed to Reactor 1 during the first 25 days of in- The bioslurry reactors incubated over a longer period of 45 days
cubation (Fig. 3). These results suggest that addition of nutrients showed further degradation of PAHs in all reactors with live cul-
in Reactor 2 may have resulted in preferential utilization of the tures. It was observed that the impact of additional nutrients in
biosurfactant amended as carbon source. Among the reactors in- Reactor 2, which caused the batch to remain behind in perfor-
oculated with the microbial culture, the one that was not supple- mance during the first 25 days (as shown by the 68.3% and 66.2%,
mented with the biosurfactant or nutrients in day 2 (Reactor 4) 2–3 ring and 4 ring PAHs removal as opposed to 80.7% and 81.6%
performed poorest. Only 30% naphthalene and 59% phenanthrene 2–3 ring and 4 ring PAHs removal in Reactor 1, Fig. 3), was not sig-
was degraded in this reactor. This reactor validated that the soil nificant after a long incubation time as Reactor 2 caught up with
 F.A. Bezza, E.M. Nkhalambayausi Chirwa / Chemosphere 144 (2016) 635–644 641

Table 2
Biodegradation of PAHs in creosote contaminated soil after incubation of the soil for 45 days; the results represent the mean ± standard deviation of the three replicates.

Compound Initial conc. mg kg−1 Final concentration in the different bioslurry reactors (mg kg−1 )

R1a Removal % R2 Removal % R3 Removal % R4 Removal %

Nap 425 ± 16 34 ± 7.4 92 ± 0.2 102 ± 4.7 76 ± 0.3 125 ± 9.1 71 ± 0.5 256 ± 17 40 ± 0.4
Ace 272 ± 11 32 ± 3.1 88 ± 0.9 32 ± 2.6 88 ± 0.7 46 ± 1.7 83 ± 0.3 74 ± 6.1 73 ± 0.3
Flou 383 ± 25 64 ± 6.7 83 ± 1.3 34 ± 4.1 91 ± 1.7 52 ± 3.6 86 ± 0.8 187 ± 11 51 ± 0.7
Phe 233 ± 3.6 5.1 ± 0.7 98 ± 1.8 31 ± 3.2 87 ± 0.9 19.5 ± 2.4 92 ± 1.4 50.7 ± 4.5 78 ± 0.1
Ant 131 ± 7.8 29 ± 3.3 78 ± 1.3 39 ± 4.2 70 ± 2.0 39.5 ± 1.6 70 ± 1.1 76 ± 3.5 42 ± 0.6

Flr 321 ± 19 31 ± 4.1 90 ± 1.9 42 ± 2.6 87 ± 0.7 37.1 ± 2.7 88 ± 0.9 136 ± 11 58 ± 0.6
Pyr 703 ± 35 82 ± 2.6 88 ± 0.4 94 ± 5.1 87 ± 0.5 106 ± 7.6 85 ± 0.7 258 ± 9.7 63 ± 0.4
BaA 145 ± 17 32 ± 1.6 78 ± 1.3 51 ± 3.2 65 ± 1.1 53.3 ± 2.4 63 ± 1.0 93 ± 7.3 36 ± 0.7
Chr 62 ± 7.8 6.9 ± 0.8 89 ± 1.4 8.4 ± 0.9 86 ± 2.3 10.7 ± 1.1 83 ± 2.2 13 ± 3.2 79 ± 2.4

BbF 57 ± 7.4 12 ± 0.6 79 ± 1.5 7.8 ± 02 86 ± 7.1 10.5 ± 0.8 82 ± 2.1 21.7 ± 2.5 62 ± 2.5
BkF 152 ± 11 33 ± 2.8 78 ± 0.9 27 ± 5.2 82 ± 3.5 51.5 ± 3.7 66 ± 0.7 45.5 ± 6.2 70 ± 0.9
BaP 24 ± 3.2 16 ± 1.1 33 ± 0.8 15 ± 2.6 38 ± 1.8 20.5 ± 2.1 15 ± 0.5 22 ± 2.7 08 ± 2.1
DahA 56 ± 07 20 ± 2.4 64 ± 1.9 15 ± 1.2 73 ± 1.6 31.4 ± 3.1 44 ± 1.1 25.1 ± 2.9 55 ± 1.8
BPer 97 ± 11 17 ± 3.4 82 ± 4.3 8.1 ± 1.6 92 ± 4.8 45 ± 2.6 54 ± 1.9 56 ± 3.7 42 ± 2.2
IcdP 3.6 ± 0.5 –b – – – – – – –

Total (mg kg−1 ) 3064.6 ± 14 414 ± 3.4 86.5 ± 1.6 506.3 ± 3.3 83 ± 2.7 648 ± 3.8 79 ± 1.2 1317 ± 7.5 57 ± 1.3

Naph, Naphthalene; Phe, Phenanthrene; Pyr, Pyrene; BbF, Benzo[b]fluoranthene; DahA, Dibenz[a,h]anthracene; Ace, Acenaphthene; Ant, Anthracene; BaA, Benz[a]anthracene;
BkF, Benzo[k]fluoranthene; BPer, Benzo[ghi]perylene; Flou, Fluorene; Flr, Fluoranthene; Chr, Chrysene; BaP, Benzo[a]pyrene; IcdP, indeno(1,2,3-cd)pyrene.
a
Reactor labels, R1 = reactor charged with biosurfactant and cells added on day 2, R2 = reactor charged with biosurfactants and nutrients NH4 NO3 and KH2 PO4 at time
zero and cells on day 2, R3 = reactor charged with nutrients only and cells added on day 2, and R4 = Control reactor with cells added on day 2 (biotic control).
b
‘––’ concentration was below instrument detection.

Fig. 4. The timeline trend of phenanthrene dissolution from the solid phase
and degradation in the liquid phase under the influence of 400 mg L−1 and
700 mg L−1 crude biosurfactant load and in the abiotic control. L-Phase represents
liquid phase and S-Phase represents solid phase.

Reactor 1 (Fig. 3). This confirms that addition of nutrient aids in

the in situ biosurfactant production following nutrient depletion
and subsequent growth limitation. Besides biosurfactant applica-
tion and nutrient biostilmulation enhanced degradations in the re-
actors can be attributed to the synergistic effects of elevated tem-
perature and slurring process. Raising the temperature increases
Fig. 3. Percentage removal of the PAHs according to their ring size in the different desorption, which makes more organic material available for mi-
reactors at day 25 (Light grey) and day 45 (Black). The results represent the mean crobial degradation. Trably and Patureau (2006) investigated the
from three independent experiments ± standard deviations. Error bars represent ability of indigenous aerobic microorganisms to degrade low and
the standard deviation of the mean of the three replicates.
high molecular mass PAHs in sewage sludge fed in continuous
bioreactors at temperature increased from 35 °C to 45 °C and 55 °C
642 F.A. Bezza, E.M. Nkhalambayausi Chirwa / Chemosphere 144 (2016) 635–644

where biodegradation of the high molecular mass PAHs was en-

hanced from 50% to 80%.

3.4. Effect of ring number on degradation

Several researchers have previously reported that PAH com-

pounds with higher ring numbers are more difficult to degrade
than low ring number compounds such as naphthalene and ace-
naphthene, for example. The low degradability of high ring num-
ber compounds has been attributed, partly, to low bioavailabil-
ity due to very low solubility limits (Xu and Obbard, 2004) and
partly due to the high metabolic energy required in multiple
ring openings which involves more complex metabolic pathways
(Kim et al., 2007). The results from the biodegradability test in
this study confirm the decreasing degradability of the compounds
with increasing ring number. The most highly biodegradable com- Fig. 5. Model fits of experimental results of Pyrene biodegradation in shake flask
pound at day 25 was phenanthrene which was 88% degraded fol- biodegradation experiments supplemented with 700 mgL−1 biosurfactant (square),
lowed by flourene, another three-ring compound at 86% degra- 400 mgL−1 biosurfactant (diamonds) and no biosurfactant (triangles).

dation. In terms of mass removed based on the amount de-

graded, Pyrene was the most degradable with a decrease from
703 to 127 mg kg−1 by day 25, a mass decrease of 576 mg kg−1 ,
Microsoft Excell 2010 (SOLVER option). Fitting the data to equation
much higher than the mass decrease of 328 mg kg−1 in flourene
(2) gave sums of squared deviations in the range of 0.035–0.0014,
(Table 1). The 5–6 rings achieved up to 81.5% removal but the
implying satisfactory fitness (Supplementary Figures S3a and b).
mass conversion was very low. All compounds were similarly af-
The values obtained for (Ce ) and their rate constants (K1 ) are pre-
fected by the addition of nutrients as shown by the decreased effi-
sented in (Supplementary Table S1) for different Lipopeptide con-
ciencies between the Reactor 1 (biosurfactant only) and Reactor 2
centrations. It can be noted that the dissolution rate increased 3
(biosurfactant + nutrients). Degradation increased further after fur-
times at 700 mg L−1 amended mirocosm than the unamended one
ther incubation to 45 days. The inhibitive effect of additional nu-
(Supplementary Table S1).
trients was less pronounced after 45 days which might either be
Pyrene biodegradation assay was run in an independent liq-
due to culture acclimation to harsher conditions or decrease in the
uid culture shake flask experiment under different concentration
toxic compounds with time resulting in the Reactor 2 performance
of biosurfactant. The experiment was conducted to evaluate the
catching up with Reactor 1.
impact of the biosurfactant on the biodegradation of Model PAH,
The biotic control (R4) had very low performance compared to
Pyrene.
the reactors supplemented with biosurfactant, nutrients, biosurfac-
PAH degradation kinetics – For a given PAH substrate, the rate
tant and nutrients at day 2. The loss from the abiotic control (R5)
of change in concentration as a result of biodegradation is mod-
is insignificant (not shown) since losses due to photodegradation,
elled as dC = qi X….. (Knights and Peters, 2006); where qi is the
volatilization and other abiotic loses were minimal. dt
biomass-normalized substrate i, utilization rate (mg substrate/mg
protein/d), Ci , is the concentration of the PAH substrate i (mg L−1 ).
3.5. PAH dissolution as rate limiting step Pyrene biodegradation kinetic parameters were determined by
assuming Monod biodegradation kinetics:
The data for mass transfer assay is obtained from an in inde-
pendent experiment conducted in shake flask bioslurry experiment
using the contaminated soil from the wood treatment plant. The dC C
= −qmax X (3)
experiment was conducted to evaluate the impact of biosurfactants dt Ks + C
on the mass transfer processes (dissolution or desorption) and sub-
sequent hydrocarbon biodegradation. Where variables are concentration of the PAH substrate C
Mass transport kinetics – Generally solubilisation involves a (mg L−1 ), biomass concentration, X (mg protein/L), and time, t(d).
series of processes: the diffusion of PAH molecules and surfac- The parameters are the maximum substrate utilization rate per
tant micelles in the solution, and the desorption of micelles at unit biomass, qmax (mg substrate/mg protein/h), the half-saturation
the PAH/water interface. The desorption model may be represented coefficient, Ks (mg/L).
by a pseudo-first-order kinetic equation described by Long et al. X, is assumed constant due to very low growth and C is the
(2014): only variable. The parameters are qmax and Ks . The integrated form
dCt of Monod equation (equation (3)) is fitted to the experimental data
− = K1 · (Ce − Ct ) (1) to yield biokinetic parameters qmax and Ks (Desai et al., 2008).
dt
The experimental data are fitted in to the Monod model and
Where Ce and Ct are the concentrations of the PAH at equilibrium the maximum substrate utilization rates per unit biomass (qmax )
and time t(d), respectively; K1 is the dissolution rate constant (d−1 ) and half saturation constant (Ks ) were determined using AQUASIM
of pseudo-first-order kinetics. The integrated form of the above software package parameter estimation technique (Reichert, 1998).
equation is: After the Monod model (equation (3)) is directly fitted to the
  set of experimental data (Fig. 5), qmax values were determined to
−Ct = Ce · 1 − e−K1 t (2)
be; 4.2; 16.1 and 19.4 mg/mg/d respectively for unamended one,
The dissolution rate constant (K1 ) and the equilibrium con- 400 mgL−1 and 700 mgL−1 amended microcosms. This shows more
centration of the PAH at time t, (Ce ) are determined by min- than 4 fold increase in the maximum substrate utilization rate per
imizing the cumulative squared residuals between experimental unit biomass of the 700 mgL−1 amended microcosm compared to
and calculated values of (Ct ) in Equation (2) using the software the unamended one.
 F.A. Bezza, E.M. Nkhalambayausi Chirwa / Chemosphere 144 (2016) 635–644 643

3.6. Significance of results Foundation (NRF) of South Africa through the Incentive Funding
for Rated Researchers Grant No. IFR2010042900080 awarded to
The results highlight the importance of dissolution rate on Prof. Evans M.N.Chirwa of the University of Pretoria. We thank Prof
biodegradability of persistent compounds in the creosote contam- Fanus Venter of the Department of Microbiology and Plant Pathol-
inated soil media. In the current study, PAH contamination of soil ogy, University of Pretoria, for his assistance with the DNA se-
was observed coming from creosote contamination of the wood quencing and characterisation of bacteria from the soil. We thank
treatment plant. The results suggest that the desorption and sub- Dr Mervyn Beukes of the Department of Biochemistry, University
sequent biodegradation of the persistent PAHs from the creosote of Pretoria, for his assistance with the protein analysis.
contaminated soil was enhanced through biosurfactant supplemen-
tation alone and simultaneous biosurfactant and nutrient supple-
Appendix A. Supplementary data
mentation. Biostimulation by addition of N and P, a strategy that
has often been reported by various studies (McKew et al., 2007)
Supplementary data related to this article can be found at http:
proved an effective approach for enhanced PAH degradation. How-
//dx.doi.org/10.1016/j.chemosphere.2015.08.027.
ever bioavailability of the PAHs can also be a limiting factor ow-
ing to their hydrophobic nature and low water solubility. As can
References
be observed in the nutrient-only amended treatment (reactor 3)
degradation of PAHs at day 45 increased to levels comparable to APHA, 2005. In: Eaton, A.D., Clesceri, L.S., Rice, E.W., Greenberg, A.E., Franson, M.A.H.
reactors amended with both nutrients and biosurfactant (reactor (Eds.). Standard Methods for the Examination of Water and Wastewater, twenty-
2), from the emulsion observed it can be evident that the PAH first ed.. American Public Health Association, American Water Works Associa-
tion, Water Environment Federation, Washington, D.C., USA (Centennial edition).
degrading community produced their own surfactants and subse- Bodour, A.A., Miller-Maier, R.M., 1998. Application of a modified drop-collapse tech-
quently hydrocarbon bioavailability was enhanced. Thus in situ bio- nique for surfactant quantitation and screening of biosurfactant-producing mi-
surfactant production through nutrient-only biostimulated treat- croorganisms. J. Microbiol. Methods 32 (3), 273–280.
Bustamante, M., Durán, N., Diez, M.C., 2012. Biosurfactants are useful tools for the
ment (introduction of additional nutrients of 11.5 mg L−1 NH4 NO3
bioremediation of contaminated soil: a review. J. Soil Sci. Plant Nutr. 12 (4),
and 1.5 mg L−1 KH2 PO4 ) resulted in considerably significant degra- 667–687.
dation and removal of the creosote PAHs as well. Just the right Chandankere, R., Yao, J., Cai, M., Masakorala, K., Jain, A.K., Choi, M.M., 2014. Prop-
erties and characterization of biosurfactant in crude oil biodegradation by bac-
amount of biosurfactant was required to kick start the culture by
terium Bacillus methylotrophicus USTB. Fuel 122, 140–148.
release of enough carbon source into solution, otherwise the cul- Chirwa, E.N., Wang, Y.T., 2000. Simultaneous Cr(VI) reduction and phenol degrada-
ture at day zero could struggle since there will not be enough tion in an anaerobic consortium of bacteria. Water Res. 34 (8), 2376–2384.
bioavailable carbon source in the system. The dynamic and the Cookson, John, T., 1995. Bioremediation Engineering: Design and Application.
McGraw-Hill, Inc..
benefit are evident from the pattern of mobilisation and degrada- Cooper, D.G., Goldenberg, B.G., 1987. Surface-active agents from two Bacillus species.
tion seen in Fig. 4. Appl. Environ. Microbiol. 53 (2), 224–229.
The technique suggested in this study could prove useful in the Desai, A.M., Autenrieth, R.L., Dimitriou-Christidis, P., McDonald, T.J., 2008. Biodegra-
dation kinetics of select polycyclic aromatic hydrocarbon (PAH) mixtures by
bioremediation of PAH contaminated soil and sediments through Sphingomonas paucimobilis EPA505. Biodegradation 19 (2), 223–233.
supplying oxygen and nutrients. The evaluated process was aero- Ghosh, I., Jasmine, J., Mukherji, S., 2014. Biodegradation of pyrene by a Pseudomonas
bic thus it supported an array of aerobic bacteria including B. sub- aeruginosa strain RS1 isolated from refinery sludge. Bioresour. Technol. 166,
548–558.
tillis and P. aeruginosa. The limitation of the process could be in the Hames, E.E., Vardar-Sukan, F., Kosaric, N., 2014. 11 patents on biosurfactants and
production of the initial biosurfactant which was complicated and future trends. Biosurfact. Prod. Util. Proc. Technol. Econ. 159, 165.
time consuming which requires either ex situ biosurfactant appli- Hazra, C., Kundu, D., Chaudhari, A., 2012. Biosurfactant-assisted bioaugmentation
in bioremediation. Microorganisms in Environmental Management. Springer,
cation or in situ biosurfactant production through supplementation
Netherlands, pp. 631–664.
of nutrients. Fortunately, once this biosurfactant production pro- Hu, J., Nakamura, J., Richardson, S.D., Aitken, M.D., 2012. Evaluating the ef-
cess has started, the rest of the process will be self-regenerating fects of bioremediation on genotoxicity of polycyclic aromatic hydrocarbon-
contaminated soil using genetically engineered, higher eukaryotic cell lines. En-
and self-sustaining, after the culture has established itself.
viron. Sci. Technol. 46 (8), 4607–4613.
Hwang, S., Cutright, T.J., 2002. Biodegradability of aged pyrene and phenanthrene in
4. Conclusion a natural soil. Chemosphere 47 (9), 891–899.
Joshi, S., Bharucha, C., Desai, A.J., 2008. Production of biosurfactant and antifungal
compound by fermented food isolate Bacillus subtilis 20B. Bioresour. Technol. 99
The results show that the biosurfactant applied twice over the (11), 4603–4608.
course of the incubation assisted the remediation by increasing the Kim, S.J., Kweon, O., Jones, R.C., Freeman, J.P., Edmondson, R.D., Cerniglia, C.E., 2007.
bioavailability of the PAHs and enhanced the degradation poten- Complete and integrated pyrene degradation pathway in Mycobacterium van-
baalenii PYR-1 based on systems biology. J. Bacteriol. 189, 464–472.
tial of the culture. With this system, enough biosurfactant could Knightes, C.D., Peters, C.A., 2006. Multisubstrate biodegradation kinetics for binary
be added to allow the cultures to grow to an effective popula- and complex mixtures of polycyclic aromatic hydrocarbons. Environ. Toxicol.
tion followed by in situ production to achieve the long-term stabil- Chem. 25 (7), 1746–1756.
Li, H., Chen, J., Jiang, L., 2014. Elevated critical micelle concentration in soil–water
ity and enhanced bioremediation of a contaminated environment. system and its implication on PAH removal and surfactant selecting. Environ.
The experiments in this study showed that in situ biosurfactant Earth Sci. 71 (9), 3991–3998.
production can be stimulated through growth limitation following Li, H., Chen, J., Wu, W., Piao, X., 2010. Distribution of polycyclic aromatic hydrocar-
bons in different size fractions of soil from a coke oven plant and its relation-
nutrient depletion. The biosurfactant produced in situ helped des- ship to organic carbon content. J. Hazard. Mater. 176 (1), 729–734.
orb and emulsify the hydrophobic contaminants and increase their Long, J., Tian, S., Niu, Y., Li, G., Ning, P., 2014. Reversible solubilization of typical
bioavailability as confirmed from the stable emulsions formed. In polycyclic aromatic hydrocarbons by a photoresponsive surfactant. Colloid. Surf.
A Physicochem. Eng. Aspects 454, 172–179.
situ biosurfactant production for bioremediation purposes is eco-
McKew, B.A., Coulon, F., Yakimov, M.M., Denaro, R., Genovese, M., Smith, C.J.,
nomically feasible and has multi-faceted advantages as it can use McGenity, T.J., 2007. Efficacy of intervention strategies for bioremediation of
the contaminant as source of carbon for biosurfactant production. crude oil in marine systems and effects on indigenous hydrocarbonoclastic bac-
teria. Environ. Microbiol. 9 (6), 1562–1571.
Melber, C., Kielhorn, J., Mangelsdorf, I., 2004. Coal Tar Creosote. Concise Interna-
Acknowledgements tional Chemical Assessment Document.
Meng, Q.X., Jiang, H.H., Hanson, L.E., Hao, J.J., 2012. Characterizing a novel strain
This research was funded through the National Research Foun- of Bacillus amyloliquefaciens BAC03 for potential biological control application.
J. Appl. Microbiol. 113 (5), 1165–1175.
dation (NRF) of South Africa through the Focus Areas Programme Reichert, P., 1998. AQUASIM 2.0 – User Manual. Swiss Federal Institute for Environ-
Grant No. CPR20110603000019146 and by the National Research mental Science and Technology, Dubendorf, Switzerland.
644 F.A. Bezza, E.M. Nkhalambayausi Chirwa / Chemosphere 144 (2016) 635–644

Shavandi, M., Mohebali, G., Haddadi, A., Shakarami, H., Nuhi, A., 2011. Emulsifica- Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular
tion potential of a newly isolated biosurfactant-producing bacterium, Rhodococ- evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30 (12), 2725–2729.
cus sp. strain TA6. Colloid. Surf. B Biointerfaces 82 (2), 477. USEPA, 1996. SW-846 Manual, Method 3550B, Ultrasonic Extraction. US Envi-
Shin, K.H., Kim, K.W., Ahn, Y., 2006. Use of biosurfactant to remediate ronmental Protection Agency http://www.epa.gov/epaoswer/hazwaste/test/pdfs/
phenanthrene-contaminated soil by the combined solubilization–biodegradation 3550b.pdf.
process. J. Hazard. Mater. 137 (3), 1831–1837. Vasileva-Tonkova, E., Sotirova, A., Galabova, D., 2011. The effect of rhamnolipid bio-
Singh, A., Van Hamme, J.D., Ward, O.P., 2007. Surfactants in microbiology and surfactant produced by Pseudomonas fluoroscens on model bacterial strains and
biotechnology: part 2. Application aspects. Biotechnol. Adv. 25 (1), 99–121. isolates from industrial wastewater. Curr. Microbiol. 62 (2), 427–433.
Szulc, A., Ambrożewicz, D., Sydow, M., Ławniczak, Ł., Piotrowska-Cyplik, A., Xia, W., Du, Z., Cui, Q., Dong, H., Wang, F., He, P., Tang, Y., 2014. Biosurfactant pro-
Marecik, R., Chrzanowski, Ł., 2014. The influence of bioaugmentation and bio- duced by novel Pseudomonas sp. WJ6 with biodegradation of n-alkanes and
surfactant addition on bioremediation efficiency of diesel-oil contaminated soil: polycyclic aromatic hydrocarbons. J. Hazard. Mater. 276, 489–498.
feasibility during field studies. J. Environ. Manag. 132, 121–128. Xu, R., Obbard, J.P., 2004. Biodegradation of polycyclic aromatic hydrocarbons in oil-
Thavasi, R., Nambaru, V.S., Jayalakshmi, S., Balasubramanian, T., Banat, I.M., 2011. contaminated beach sediments treated with nutrient amendments. J. Environ.
Biosurfactant production by Pseudomonas aeruginosa from renewable resources. Qual. 33 (3), 861–867.
Indian J. Microbiol. 51 (1), 30–36. Zhang, Y.I.M.I.N., Miller, R.M., 1992. Enhanced octadecane dispersion and biodegra-
Tikilili, P.V., Chirwa, E.M.N., 2011. Characterization and biodegradation of polycyclic dation by a Pseudomonas rhamnolipid surfactant (biosurfactant). Appl. Environ.
aromatic hydrocarbons in radioactive wastewater. J. Hazard. Mater. 192 (3), Microbiol. 58 (10), 3276–3282.
1589–1596. Zhu, H., Aitken, M.D., 2010. Surfactant-enhanced desorption and biodegradation of
Trably, E., Patureau, D., 2006. Successful treatment of low PAH-contaminated sewage polycyclic aromatic hydrocarbons in contaminated soil. Environ. Sci. Technol. 44
sludge in aerobic bioreactors. Environ. Sci. Pollut. Res. 13 (3), 170–176. (19), 7260–7265.
Trummler, K., Effenberger, F., Syldatk, C., 2003. An integrated microbial/enzymatic
process for production of rhamnolipids and L- (+)-rhamnose from rapeseed oil
with Pseudomonas sp. DSM 2874. Eur. J. lipid Sci. Technol. 105 (10), 563–571.