International Biodeterioration & Biodegradation 106 (2016) 106e116

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier.com/locate/ibiod

Degradation of crude oil and relationship with bacteria and enzymatic

activities in laboratory testing*
Yongrui Pi a, b, Long Meng a, b, Mutai Bao a, b, *, Peiyan Sun c, Jinren Lu b
a
Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100, China
b
College of Chemistry & Chemical Engineering, Ocean University of China, Qingdao 266100, China
c
Key Laboratory of Marine Spill Oil Identification and Damage Assessment Technology, North China Sea Environmental Monitoring Center, State Oceanic
Administration, Qingdao 266033, China

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

Article history: The biodegradation of petroleum hydrocarbons is one of the most important processes involved in the
Received 31 August 2015 weathering and eventual removal of petroleum hydrocarbons from the marine environment. The effect
Received in revised form of four variablesd(NH4)2SO4, K2HPO4, temperature and inoculationdon crude oil biodegradation (BDR)
20 October 2015
were evaluated using response surface methodology to confirm the nutrients effect on the biodegra-
Accepted 20 October 2015
Available online xxx
dation and explore the relationships between BDR and bacterial biomass, dehydrogenase activity and
peroxidase activity. These variables were optimized to allow the highest removal of crude oil, and the
results indicated that crude oil removal could increase to even higher levels if the temperature and the
Keywords:
Crude oil
concentrations of (NH4)2SO4 and K2HPO4 were increased. Following biodegradation, the enzymatic ac-
Biodegradation tivities were evaluated using a modified spectrophotometric method; the results showed that an in-
Response surface methodology crease in the temperature and inoculation quantity resulted in a higher dehydrogenase activity (mg TPF
The relationship (l$h)
1) and that the highest peroxidase activity (18.50 U) occurred at 2.01 g$l
1 (NH4)2SO4,
Enzymatic activities 1.10 g$l
1 K2HPO4, 25  C and 1.0% inoculation. The crude oil BDR increased substantially with bacterial
Bacterial biomass biomass and decreased slightly with dehydrogenase activity or peroxidase activity, which was consistent
with the coefficients in the fitted equation. The correlation between microbial degradation of crude oil
and bacterial biomass and enzymatic activities can be used in simulations of biodegradation processes
with petroleum hydrocarbons, PAHs and other hydrocarbon compounds and provides a more thorough
understanding of the microbial community's function in contaminated soil.
© 2015 Published by Elsevier Ltd.

1. Introduction Rosenberg, 2014). Thus, microbial oil biodegradation is recog-

nized as one of the most important methods for petroleum hy-
The petroleum hydrocarbons in crude oils, such as those drocarbons remediation (Mahmoudi et al., 2013; McGenity, 2014;
released into marine ecosystems by the Exxon Valdez and the BP Silliman et al., 2012).
Deepwater Horizon spills, may move hydrocarbons into other Most petroleum hydrocarbons are biodegradable under aerobic
environmental compartments through physical and chemical pro- (Cajal-Marin~ osa et al., 2012; Tahhan et al., 2011) and anaerobic
cesses, such as shoreline erosion, volatilization, water washing, or conditions (Mbadinga et al., 2011; Li et al., 2012; Wang et al., 2011).
the addition of dispersants; however, these processes do not reduce Hydrocarbon-oxidizing bacteria capable of growth on aliphatic and
the toxicity of these compounds. Biodegradation mediated by aromatic hydrocarbons are found in many genera. In the presence
indigenous microbial communities is a key process by which pe- of O2, the initial steps in the bacterial degradation of hydrocarbons
troleum hydrocarbons are mineralized and removed from rely on oxygenases. These oxygenases are membrane-bound, the
contaminated environments (Atlas and Hazen, 2011; Ron and cell must come into direct contact with their water-insoluble sub-
strates. Because the oxygenases are group-specificdfor example,
some degrade specific fractions of alkanes, whereas others work on
*
This is MCTL Contribution No. 26. aromatics or cyclic hydrocarbonsdit follows that only a mixture of
* Corresponding author. Key Laboratory of Marine Chemistry Theory and Tech- different microorganisms can efficiently degrade crude oil and
nology, Ministry of Education, Ocean University of China, Qingdao 266100, China
petroleum fractions. The polycyclic aromatic hydrocarbons (PAHs)
E-mail address: [email protected] (M. Bao).

http://dx.doi.org/10.1016/j.ibiod.2015.10.015
0964-8305/© 2015 Published by Elsevier Ltd.
 Y. Pi et al. / International Biodeterioration & Biodegradation 106 (2016) 106e116 107

are a minor constituent of crude oils; however, they are among the applications. The present study employed RSM to analyze the
most toxic to plants and animals. Bacteria can completely convert enzymatic activities and the effects of enzymatic activities on the
PAHs to biomass, CO2, and H2O, but they usually require the initial biodegradation of crude oil.
insertion of O2 via dioxygenase enzymes. Wang et al. (2011)
employed an alkane-degrading methanogenic enrichment culture 2. Materials and method
to understand the function of microbial communities in oil reser-
voirs under anaerobic conditions and indicated that the archeal 2.1. Sampling and microorganism acclimatization
phylotypes were mostly related to members of the orders Meth-
anobacteriales and Methanosarcinales. Li et al. (2012) investigated Seawater and sediments were collected from a beach in Jiaoz-
the microbial community characteristics of petroleum reservoir hou Bay, Qingdao (Loushan River, 36120 N, 120 200 E). After delivery
production water amended with n-alkanes and incubated under to the laboratory, the samples were filtered and stored along with
nitrate-, sulfate-reducing and methanogenic conditions, and the soil samples in a refrigerator at 4  C.
phylogenetic analyses based on 16S rRNA gene amplification indi- The enrichment medium contained 3.0 g of beef extract, 10 g of
cated that a-, b-, g-Proteobacteria and Bacteroidetes were detected peptone, and 5.0 g of NaCl per liter. The basic medium used for
in the nitrate-reducing enrichment; Actinobacteria, Nitrospira and screening was mineral salt medium (MSM) with crude oil as the
d-Proteobacteria were recovered from both the sulfate-reducing and sole carbon source. The MSM contained 0.5 g of K2HPO4, 2.0 g of
methanogenic enrichments. Mbadinga et al. (2012) reviewed the NaSO4, 1.0 g of NH4Cl, 0.02 g of MgSO4$7H2O, 0.07 g of CaCl2
anaerobic biodegradability of alkanes in terms of the microorgan- (Christian et al., 2009) and 1.0 ml of trace salt solution per liter. The
isms involved and the biochemical pathways. Diverse physiological trace salt solution was defined as 30 mg of FeCl3, 0.5 mg of CuSO4,
groups of microorganisms (isolates or consortia) such as Thermo- 0.5 mg of MnSO4$H2O, and 10 mg of ZnSO4$7H2O per liter. The pH
coccus, Firmicutes and d-Proteobacteria, grown under chlorate- was adjusted to 7.0e7.2 with 1.0 M NaOH and 1.0 M HCl prior to
reducing, nitrate-reducing, sufidogenic or methanogenic condi- sterilization.
tions can use alkanes as carbon and energy sources. Most inter- Hydrocarbon-degrading bacteria present in the soil and water
estingly, Ettwig et al. (2010) proposed a pathway to produce samples were isolated in two ways: (a) by direct cultivating of di-
oxygen, indicating that bacterium named ‘M. oxyfera’ could bypass lutions of the samples on mineral salt medium containing crude oil
the denitrification intermediate nitrous oxide by the conversion of as the sole carbon and energy sources; and (b) by plating of
two nitric oxide molecules to dinitrogen and oxygen under anoxic enrichment cultures of the samples prepared in mineral salt broth,
conditions. also containing crude oil as the sole carbon and energy sources.
Enzymes play an important role in the microbial degradation of
oil, chlorinated hydrocarbons, fuel additives, and many other 2.2. Box-Behnken design for response surface methodology
compounds (Atlas, 1991; Das and Chandran, 2010; Van Beilen and
Funhoff, 2007). Monooxygenases and dioxygenases, which are The Box-Behnken design for response surface methodology was
produced by hydrocarbon-degrading bacteria, can catalyze the employed using Design-Expert Software Version 9. The experi-
initial oxidation reactions of n-alkane and aromatic hydrocarbons mental design consisted of four factors: sodium nitrate concen-
to primary alcohols (monooxygenase reactions with hydroxylases) tration (nitrogen source), dipotassium phosphate concentration
and trans-dihydrodiols (monooxygenase reactions) or cis-dihy- (phosphorous source), temperature and inoculation quantity. Other
drodiols (dioxygenase reactions). Further oxidation of the trans- cultivation parameters were held constant. All of the experiments
dihydrodiols and the cis-dihydrodiols leads to the formation of were performed in 250-ml flasks. Table 1 depicts the three levels of
catechols, which are substrates for other dioxygenases that catalyze variables for the selected factors. In the experimental design, 29
enzymatic cleavage of the aromatic ring (Das and Chandran, 2010; experiments with varying nutrient combinations and five repli-
Haritash and Kaushik, 2009; Juhasz and Naidu, 2000). The alkane cates at the central points were carried out. The effect of inde-
hydroxylases play a key role in the special oxidation of the CeH pendent factors on the dependent factors was analyzed using a
bond cleavage. Van Beilen and Funhoff (2007) reviewed long-chain quadratic equation:
n-alkane oxidation for two classes of enzymes: (a) the class of cy-
tochrome P450-related enzymes in both yeasts and bacteria, e.g., X
3 X
3 X
3 X
3
bacterial CYP153 enzymes, and (b) the class of bacterial particulate Y ¼ a0 þ ai xi þ aii x2i þ aij xi xj (1)
alkane hydroxylases. Wentzel et al. (2007) summarized the bacte- i¼1 i¼1 i¼1 i < j¼2
rial metabolism of long-chain n-alkanes and confirmed that
enzyme systems involved in the utilization of long-chain n-alkanes where Y is the response, a0 is the offset term, ai is the ith linear
have led to an improved understanding of microbial long-chain n- coefficient, aii is the squared term coefficient, and aij is the ijth
alkane metabolism. interaction coefficient.
Response surface methodology (RSM) is a useful mathematical
and statistical method for analyzing the relation between several 2.3. Biodegradation of crude oil experimentation
independent variables and one or more responses. The method,
which was developed by Box and Wilson (1951), has been applied Batches of media sufficient for each product (200 ml) were
in many fields of science, such as heavy metal biosorption (Alslaibi prepared and dispensed into 250-ml flasks prior to sterilization.
et al., 2013; Şener et al., 2014; Zolgharnein et al., 2013), biodiesel The LSH bacteria (2.0%, v/v) which were isolated from Loushan
production (Karpagam et al., 2015; Maran and Priya, 2015) and lipid River were added into the MSM with 0.5% (w/v) crude oil. The
production (Hallenbeck et al., 2014; Singh et al., 2015). Recently, culture conditions were maintained as described in the previous
RSM has been used in bioremediation of oil spills (Bravo-Linares section. The remaining oil was extracted twice from the culture
et al., 2013; Zahed et al., 2012), enhanced chrysene degradation fluid with 50 ml of petroleum ether, and the petroleum ether phase
by halotolerant Achromobacter xylosoxidans (Ghevariya et al., 2011), was then collected 20 min after extraction. The organic phase was
biodegradation of n-alkanes from weathered crude oil in coastal subsequently analyzed by UV spectrophotometry. All treatments
sediments (Mohajeri et al., 2011), degradation of phenols except the sterile control were performed in triplicate. The aqueous
(Annadurai et al., 2008; Yahiaoui et al., 2012), and other fraction was used for next step.
 108
Table 1
Box-Behnken design with actual responses for biodegradation removal (%), bacterial density (1010 cells/mL), dehydrogenase activity (mg TPF/L$h) and peroxidase activity (U) with predicted responses after analysis.

Runs Factor A Factor B Factor C Factor D Observed responses Predicted responses

(NH4)2SO4 K2HPO4 (g/ temperature inoculation
Biodegradation Bacterial density Dehydrogenase Peroxidase Biodegradation Bacterial density Dehydrogenase Peroxidase
(g/L) L) ( C) quantity (%)
removal (%) (Y1) (1010 cells/mL) (Y2) activity(mg TPF/L$h) removal (%) (Y1) (1010 cells/mL) (Y2) activity(mg TPF/L$h)

Y. Pi et al. / International Biodeterioration & Biodegradation 106 (2016) 106e116

activity (U) activity (U)
(Y3) (Y4) (Y3) (Y4)

1 2.02 1.55 25 3 38.56 11.85 39.31 12.97 39.21 11.58 36.95 9.64
2 3.01 1.50 25 1 39.17 15.26 11.74 6.82 37.98 11.80 50.23 4.77
3 2.00 1.50 25 3 31.18 8.67 28.37 10.29 28.46 7.71 37.97 6.65
4 2.00 1.60 25 3 29.78 8.23 36.66 15.65 32.38 10.52 46.60 8.30
5 3.03 1.05 25 3 35.81 6.60 29.86 1.91 37.12 5.34 39.45 4.26
6 1.01 1.55 20 3 37.10 9.87 37.82 5.42 35.26 10.66 42.13 7.66
7 2.01 1.55 20 5 34.52 11.81 67.13 3.33 37.15 10.43 44.19 5.30
8 1.03 1.55 25 1 34.98 14.61 53.78 3.55 35.91 12.36 29.94 3.98
9 1.03 2.00 25 3 42.11 11.99 59.54 0.76 39.24 9.87 51.95
0.09
10 2.01 1.05 25 5 30.04 8.99 62.18 13.70 31.08 7.53 52.79 10.32
11 1.04 1.60 30 3 36.50 11.92 43.92 4.53 37.12 11.45 50.84 6.65
12 2.02 2.05 20 3 20.16 11.97 85.44 1.34 28.08 14.21 50.23 6.72
13 3.02 1.55 20 3 32.42 5.52 26.99 9.17 35.34 10.80 26.72 4.61
14 2.03 1.05 30 3 38.53 9.22 9.02 14.07 34.98 10.40 33.47 16.15
15 2.04 1.00 20 3 36.38 10.65 37.18 7.52 37.12 14.00 48.12 6.14
16 2.01 2.05 30 3 40.87 11.68 35.25 5.96 42.85 14.21 31.55 4.90
17 2.02 2.00 25 1 43.10 14.76 6.86 4.54 37.12 14.21 30.86 6.50
18 2.05 1.50 20 1 32.19 7.66 57.68 5.13 40.26 10.44 56.51 5.91
19 3.05 1.50 25 5 42.01 13.11 18.06 2.63 35.69 13.99 16.29 6.65
20 3.04 2.05 25 3 45.82 14.49 40.94 6.55 40.89 12.90 33.80 8.74
21 2.04 1.50 25 3 38.37 11.53 54.45 9.03 34.79 9.09 57.39 8.98
22 2.01 1.10 25 1 38.51 9.07 18.84 18.50 41.72 10.00 26.11 13.41
23 3.04 1.50 30 3 41.25 11.21 36.70 5.65 37.40 10.08 14.37 10.10
24 1.01 1.50 25 5 38.68 18.02 93.17 9.49 37.98 14.21 50.23 7.31
25 2.00 1.60 30 1 34.91 9.61 32.35 8.16 35.18 10.19 50.23 11.30
26 1.03 1.00 25 3 33.80 7.54 4.30 7.84 37.12 8.56 32.58 6.65
27 2.01 1.60 30 5 34.67 14.64 47.45 4.39 36.72 14.21 26.42 7.93
28 2.03 1.55 25 3 38.18 5.03 28.44 7.97 32.86 8.06 50.23 6.65
29 2.02 2.05 25 5 33.62 17.40 87.00 2.92 36.22 14.10 72.29 3.68
 Y. Pi et al. / International Biodeterioration & Biodegradation 106 (2016) 106e116 109

2.4. Analysis of enzymatic activities removal efficiencies were found to decrease when moving away
from the point, indicating that either an increase or decrease in any
The above aqueous fraction after extracting oil was centrifuged of the tested variables resulted in a decline in the response.
at 8000 rpm and cleaned with 0.9% physiological saline more than When the effect of different concentrations of (NH4)2SO4 and
three times. Then the cells in the centrifuge tube were suspended K2HPO4 on crude oil BDR are examined (Fig. 1a), it can be seen that
with 0.9% physiological saline, measured the absorption spectro- the crude oil BDR increases with (NH4)2SO4 and K2HPO4 concen-
photometry of the bacteria suspension at l ¼ 600, until OD600 ¼ 2.0. trations, particularly at high levels of K2HPO4. The effect of
The measurement of dehydrogenase activity was performed using (NH4)2SO4 concentration and temperature on crude oil BDR was
a modified spectrophotometric method described by Wu et al. also examined (Fig. 1b). Interestingly, from these curves, it can be
(2014). A 2-ml aliquot of the OD600 ¼ 2.0 bacterial suspension deduced that at a given concentration of (NH4)2SO4, the crude oil
was treated with a solution of 2 ml of 1.0 g l
1 2,3,5- BDR is dependent on temperature, at least within the range
triphenyltetrazolium chloride (TTC)-glucose in 2 ml of 0.1 M Tris examined here. It is also evident from Fig. 1c that the crude oil BDR
buffer solution (pH 7.6) and was ultrasound incubated for 1 h at is independent of (NH4)2SO4 concentration when the inoculation
37  C. After the reduced triphenylformazan (TPF) formed, it was quantity is decreased. Fig. 1d shows that the crude oil BDR increases
extracted with 5 ml of ethyl acetate and was quantified spectro- with K2HPO4 concentration and temperature. From Fig. 1e, we can
photometrically at 485 nm. All experiments were conducted in see that compared with the inoculation quantity, the K2HPO4
triplicate, and the activity was expressed as mg TPF (L$h)
1. concentration has a greater effect on the crude oil BDR. Finally,
Peroxidase activity was measured as the rate of substrate Fig. 1f demonstrates the effect of different inoculation quantities
oxidation in the presence of added H2O2 (Bach et al., 2013). A 2-ml and temperatures on crude oil BDR, again showing that inoculation
aliquot of the OD600 ¼ 2.0 bacterial suspension was treated with a quantity has a very little influence and temperature has a strong
solution of 1.5 ml of 0.2 mol l
1 phosphate buffer (pH ¼ 6.0) and effect. The influence of temperature is greatest near the optimal
ultrasound incubated for 30 min at 40  C in a 10-ml test tube. values of (NH4)2SO4, K2HPO4 and inoculation quantity.
Following this incubation, 0.5 ml of 0.3% H2O2 and 1 ml of
0.05 mol l
1 guaiacol solution were added to the test tube, blended 3.2. Interaction among factors influencing bacterial biomass
rapidly, shocked for 3 s, and then immediately poured into a
colorimetric tube. The mixtures were quantified spectrophoto- It is well known that the bacterial biomass is always limited by
metrically at 470 nm every minute for 5 min. The 0.01 per minute the bioavailability of nutrients such as nitrogen and phosphorus
A470 changes were recorded as a unit of peroxidase activity (U). and environmental factors such as temperature. Based on the BBD
analysis, a model (Eq. (3)) was derived that could relate bacterial
biomass as a measured output in response to the independent
3. Results input variables. Analysis of variance (ANOVA) was used to assess
the significance of each variable in the model. Multiple regression
3.1. Interaction among factors influencing crude oil biodegradation analysis was used to analyze the bacterial density (BD)
(1010 cells$ml
1); the polynomial equation derived from the
The optimal combination of the four factors, nitrate concentra- regression analysis was as follows:
tion (nitrogen source), dipotassium phosphate concentration
(phosphorous source), temperature and inoculation quantity was BD ¼ 34:2883
3:3549  A þ 2:5248  B
2:4150  C
determined through a Box-Behnken design (BBD). The reasonable
agreement between the response values obtained by experimen-
þ 7:6438  D þ 5:2850  AB
1:3838  AD
1:0012
tation and the predicted values of the design expert software  A2
2:1097  B2
(Table 1) indicated that the critical factors of the system could be
(3)
described by a quadratic equation. The BBD design yielded a
maximum crude oil removal that was further validated. The opti- where A is the concentration of (NH4)2SO4 (g$l
1), B is the concen-
mization process for the overall maximum biodegradation per- tration of K2HPO4 (g$l
1), C is the temperature ( C), and D is the
centage (BDRP) was formulated as a quadratic function (Eq. (2)): inoculation quantity (%). The maximum BD (1010 cells$ml
1) can be
predicted by varying the concentrations of (NH4)2SO4 (g$l
1) and
BDRP ¼
40:5885 þ 28:0258  A þ 13:9642  B þ 1:2914  C
K2HPO4 (g$l
1), the temperature ( C) and the inoculation quantity (%)
þ 11:7879  D þ 3:5950  AB
0:8505  AC in the above equation. Design-Expert® software allows for numeric

2:1975  AD
1:2480  BC
0:9325  BD optimization of the assessed variables, such that the input variables
can either be set at maximum, minimum, or target values within a

1:0483  A2 þ 4:7767  B2 range. In this part of the analysis, the response was set to maximize
(2) bacterial density and the resultant optimal responses for (NH4)2SO4,
K2HPO4, temperature and inoculation quantity were found to be 2.0,
where A is the concentration of (NH4)2SO4 (g$l
1), B is the con- 1.5, 30  C and 5%, respectively, with a maximum bacterial density of
centration of K2HPO4 (g$l
1), C is the temperature ( C), and D is the 18.1  1010 cells$ml
1. Fig. 2 shows that the 3D graph reflects the
inoculation (%). The maximum BDRP (%) can be predicted by interaction of the four factors with the response of the bacterial
varying the concentrations of (NH4)2SO4 (g$l
1) and K2HPO4 biomass. Based on this information, a contour plot was generated for
(g$l
1), the temperature ( C) and the inoculation (%) in the above pairwise combinations of (NH4)2SO4 and K2HPO4 (Fig. 2a), temper-
equation. A positive sign corresponds to a synergistic effect in the ature and (NH4)2SO4 (Fig. 2b), (NH4)2SO4 and inoculation quantity
response, whereas a negative sign corresponds to an antagonistic (Fig. 2c), temperature and K2HPO4 (Fig. 2d), K2HPO4 and inoculation
effect. The 3D graphs reflect the interaction of the four factors with quantity (Fig. 2e), and temperature and inoculation quantity (Fig. 2f).
the response of the BDRP of crude oil (Fig. 1). From the figure, it is From the figure, it can be seen that for the high 3D area, the optimum
possible to see that for the high 3D area, the optimum removal bacterial density of 18.0  1010 cells$ml
1 occurred at approximately
percentage of 45.8% occurred at approximately 3.0 g l
1 (NH4)2SO4, 1.0 g l
1 (NH4)2SO4, 1.5 g l
1 K2HPO4, 25  C and 5.0% inoculation
2.0 g l
1 K2HPO4, 25  C and 3.0% inoculation. The biodegradation quantity.
110 Y. Pi et al. / International Biodeterioration & Biodegradation 106 (2016) 106e116

Fig. 1. 3D surface plot for the response of biodegradation removal (%) under different concentrations of (NH4)2SO4 (1.0e3.0 g/L), K2HPO4 (1.0e2.0 g/L) and inoculation quantity (1%e
5%) at different temperatures (20  Ce30  C).

3.3. Enzymatic activities after biodegradation

PA ¼ 82:1900
29:2242  A
37:0550  B
0:9241  C
The relations between the independent variables and dehy-
drogenase activity (DA) and peroxidase activity (PA) exhibited a
7:2683  D þ 4:1650  AB þ 0:4205  AC þ 3:2625
quadratic function that was developed through multiple regression  AD þ 1:2960  BC þ 0:8800  BD0:8967  B2 þ 0:6846
analysis of the experimental data; the final models acquired are
given below:  D2
(5)
DA ¼ 733:2699
12:4617  A
210:0283  B
36:8381  C

48:6527  D
4:6850  AB þ 1:6815  AC
4:6550 where A is the concentration of (NH4)2SO4 (g$l
1), B is the con-
centration of K2HPO4 (g$l
1), C is the temperature ( C), and D is the
 AD þ 1:7350  BC þ 1:9500  BD þ 2:7608  CD inoculation quantity (%).

2:0383A2 þ 59:5767B2
2:0255  D2 The 3D RSM plots of the four parameters were constructed for
dehydrogenase activity (mg TPF (L$h)
1) (Fig. 3) and peroxidase
(4)
activity (U) (Fig. 4) and were plotted as a function of two of the
 Y. Pi et al. / International Biodeterioration & Biodegradation 106 (2016) 106e116 111

Fig. 2. 3D surface plot for the response of bacterial density (1010 cells/mL) under different concentrations of (NH4)2SO4 (1.0e3.0 g/L) and K2HPO4 (1.0e2.0 g/L) and inoculation
quantity (1%e5%) at different temperatures (20  Ce30  C).

factors while the others were held constant at their mean values. 3.4. Perturbation analyses
The interaction between the corresponding variables was
negligible when the contour of the response surface was circular. Perturbation analysis was carried out using the previous re-
It can be seen from Fig. 3 that an increase in temperature and sults (Fig. 5); this type of plot shows the effect on the response
inoculation quantity results in higher dehydrogenase activity when one factor varies while the other factors are held constant.
(mg TPF (L$h)
1), which may be attributed to the increase in In this case, it is readily apparent that different (NH4)2SO4 con-
bacterial biomass; consequently, the product of dehydrogenase centrations (Fig.5a, trace A) have very little effect on BDR.
activity trended upward. Fig. 4 shows that the highest peroxi- However, BDR is responsive to K2HPO4. Additionally, inoculation
dase activity (18.50 U) occurred at 2.01 g l
1 (NH4)2SO4, quantity plays a key role in determining the bacterial biomass,
1.10 g l
1 K2HPO4, 25  C and 1.0% inoculation quantity. The which increased monotonically as that factor was increased
decrease observed away from these values can be attributed to throughout the range examined (Fig.5b, trace D). Finally, this
peroxidase activity. analysis shows that dehydrogenase activity (mg TPF (L$h)
1) and
112 Y. Pi et al. / International Biodeterioration & Biodegradation 106 (2016) 106e116

Fig. 3. 3D surface plot for the response of dehydrogenase activity (mg TPF/L$h) under different concentrations of (NH4)2SO4 (1.0e3.0 g/L) and K2HPO4 (1.0e2.0 g/L) and inoculation
quantity (1%e5%) at different temperatures (20  Ce30  C).

peroxidase activity (U) are quite sensitive to inoculation quantity

as seen from the steepness of the response curve (Fig.5c, d; trace Y ¼ 33:0525 þ 0:8225X1
0:1339X2
0:0547X3 (6)
D). Temperature and K2HPO4 are much more responsive to the
enzymes' activity. where Y is biodegradation removal (%), X1 is bacterial density
(1010 cells$ml
1), X2 is dehydrogenase activity (mg TPF (L$h)
1),
and X3 is peroxidase activity (U). Compared with the three factors in
3.5. Correlation between biodegradation removal and bacterial the equation above, bacterial density plays the most significant and
biomass, dehydrogenase activity and peroxidase activity positive role in the degradation of crude oil. The 3D images in Fig. 6
clearly demonstrate that the crude oil BDR increased strongly with
The correlation between biodegradation removal and bacterial bacterial biomass and decreased slightly with dehydrogenase ac-
biomass, dehydrogenase activity and peroxidase activity was fitted tivity or peroxidase activity, which was consistent with the co-
with a ternary linear equation using Matlab software. The equation efficients in the equation. While the negative effect of
is as follows: dehydrogenase activity or peroxidase activity on the
 Y. Pi et al. / International Biodeterioration & Biodegradation 106 (2016) 106e116 113

Fig. 4. 3D surface plot for the response of peroxidase activity (U) under different concentrations of (NH4)2SO4 (1.0e3.0 g/L) and K2HPO4 (1.0e2.0 g/L) and inoculation quantity (1%e
5%) at different temperatures (20  Ce30  C).

biodegradation removal of crude oil is very interesting, it could be while bioaugmentation had no additional effect. Bravo-Linares
explained if some of the proteins secreted by the microbes could et al. (2013) investigated different factors that were important at
inhibit the growth of other microorganisms. different intertidal locations using RSM, and the results indicated
that at low tide, the timing of application was most important,
4. Discussion whereas at the mid-tide location, the proportion of biosolvent was
most important. Of the nutrient additions, inorganic nutrients
Petroleum hydrocarbons degradation in the marine environ- alone were more effective than organic forms of nitrogen or mix-
ment is typically limited by the bioavailability of nutrients such as tures of urea with inorganic nutrients. Zahed et al. (2012) studied
nitrogen and phosphorus, organisms, environmental factors such the bioremediation of dispersed crude oil using RSM based on ni-
as temperature, and ecological interactions (Head et al., 2006). trogen and phosphorus supplementation in a closed system; the
Kauppi et al. (2011) compared means to stimulate biodegradation highest dispersed crude oil removal (68.1%) was achieved at 28
of diesel oil hydrocarbons in contaminated soil, and concluded that days with a phosphorus concentration of 1.2 mg l
1. The optimum
biostimulation via optimization of nitrogen and oxygen supply removal (45.8%) in our research was at 7 days and is almost twice
significantly improved bioremediation of oil-contaminated soil, the value reported (24.3%) for the same amount of time.
114 Y. Pi et al. / International Biodeterioration & Biodegradation 106 (2016) 106e116

Perturbation Perturbation
b
a

B B

D D
B
BDR

CD

BD
A C
C A A D
C A
B

Deviation from Reference Point (Coded Units) Deviation from Reference Point (Coded Units)

Perturbation Perturbation
d
c

B D
PA
DA

B C C
A B
D
C
A D A A
D C
B

Deviation from Reference Point (Coded Units) Deviation from Reference Point (Coded Units)

Fig. 5. Perturbation analyses. (a) Biodegradation removal (%), (b) bacterial density (1010 cells/mL), (c) dehydrogenase activity (mg TPF/L$h), and (d) peroxidase activity (U) as a
function of changes in factors.

Das and Chandran (2010) reported that enzymes participating in bioremediation of diesel oil-contaminated soil and demonstrated
the degradation of hydrocarbons play an important role in the that microbial counts, soil respiration and enzyme activities were
microbial degradation of oil, chlorinated hydrocarbons, fuel addi- significantly positively correlated with one another and with the
tives, and many other compounds. Typical peroxidases produce residual hydrocarbon concentration. Margesin et al. (2003) indi-
organic radicals through single-electron extractions from organic cated that there is no correlation between the prevalence of
substrates. The radicals released can undergo various subsequent hydrocarbon-degradative genotypes and biological activities
spontaneous reactions, including ether cleavage in dioxins (Valli (respiration, fluorescein diacetate hydrolysis, lipase activity) and
et al., 1992), quinone formation from PAHs and chlorophenols the numbers of cultivable hydrocarbon-degrading microorgan-
(Majcherczyk et al., 1998; Reddy et al., 1998) and the abiotic isms; there was also no correlation between the number of hy-
oxidative coupling of EDCs (nonylphenol and bisphenol A) or PAHs drocarbon degraders and the contamination level. Ting et al.
(Cabana et al., 2007; Junghanns et al., 2005). Ma et al. (2015) (2011) studied biodegradation of phenanthrene and pyrene by a
demonstrated that the high degradation efficiency during treat- white rot fungus, Ganoderma lucidum, and found that the biomass
ment may correlate with dehydrogenase activity and an increase in of the organism decreased with the increase of PAH concentration
the microbial population, particularly the population of functional in the cultures. Tahhan et al. (2012) investigated the effect of
microbes. Phenylphosphate carboxylase is the key enzyme involved successive inoculation with hydrocarbon-degrading bacteria on
in anaerobic phenol degradation, which is formed from phenol by the dynamics of petroleum hydrocarbons degradation in soil, and
ATP-dependent phenylphosphate (Narmandakh et al., 2006; confirmed carbon mineralization was evaluated by respirometers
Schmeling et al., 2004). Guo et al. (2010) investigated the initial while total petroleum hydrocarbons and its fractions were gravi-
dioxygenase genes of 21 PAH-degrading bacteria isolated from metrically evaluated by extraction from soil and fractionation.
mangrove sediments, most of the isolates belonged to the genera of Ziervogel et al. (2012) demonstrated high rates of enzymatic ac-
Sphingomonas and Mycobacterium, and the other included Rhodo- tivity (lipase hydrolysis) indicative of oil degradation in oil-
coccus, Paracoccus and Pseudomonas. contaminated surface seawater, and the results showed
Some correlations during the biodegradation of petroleum enhanced microbial activities that were not directly associated
hydrocarbon compounds have been reported in the relevant with primary oil degradation (peptidase), as well as a twofold
literature. Margesin and Schinner (2001) investigated the increase in DOC. Huang et al. (2012) studied the correlation among
 Y. Pi et al. / International Biodeterioration & Biodegradation 106 (2016) 106e116 115

Fig. 6. The correlation between biodegradation removal and bacteria biomass, dehydrogenase activity and peroxidase activity.

soil microorganisms, soil enzyme activities, and removal rates of Conflicts of interest
pollutants in three constructed wetlands and demonstrated that
there were good linear relationships between the canonical vari- No conflict of interest exits in the submission of this manuscript,
ables of microbes and enzymes and that soil enzyme activities had and the manuscript is approved by all authors for publication. I
more significant associations with the NH4þ-N removal rate than would like to declare on behalf of my co-authors that the work
the soil microorganisms. All of these studies provide crucial in- described was original research that has not been published pre-
formation on the bioremediation process in spill oil-contaminated viously and is not under consideration for publication elsewhere, in
environments. Santos et al. (2012) observed that measured soil whole or in part. All of the authors listed have approved the
enzyme activities correlated well with microbial population levels, enclosed manuscript.
community structures and rates of respiration (CO2 production);
relatively simple enzyme assays can be used to efficiently evaluate Acknowledgments
the metabolic activity of soil microbial communities under crude
oil contamination. The results in the present study are consistent This research was funded by the National Natural Science
with the results reported above. The correlation between Foundation of China (41376084), the Major Projects of National
biodegradation removal and bacterial biomass, dehydrogenase High Technology Research and Development Program 863
activity and peroxidase activity was studied using Matlab soft- (2013AA064401), the Public Science and Technology Research
ware. The crude oil BDR increased strongly with bacterial biomass Funds Projects of Ocean (201205012), the Program for New Century
and decreased slightly with dehydrogenase activity or peroxidase Excellent Talents in University (NCET-11-0464), the Program for
activity, which was consistent with the coefficients in the equa- Innovative Research Team in University (IRT1289), and the Open
tion. This correlation could be employed in the modeling of field Foundation of Key Laboratory of Marine Spill Oil Identification and
bioremediation of oil-contaminated environments. Damage Assessment Technology of SOA (201402).
116 Y. Pi et al. / International Biodeterioration & Biodegradation 106 (2016) 106e116

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