The Science of the Total Environment, 52 (1986) 109--121 Elsevier Science Publishers B.V., Amsterdam --Pr in ted in The Netherlands
109
BIODEGRADATION OF USED MOTOR OIL BY BACTERIA PROMOTES THE SOLUBILIZATION OF HEAVY METALS
RAFAEL VAZQUEZ-DUHALT and HUBERT GREPPIN
D~partment de Biologie v~g~tale, Universit~ de Gen$ve, 3, place de l'Universit$, 1211 Gen~ve 4 (Switzerland)
(Received September 2nd, 1985; accepted September 12th, 1985)
ABSTRACT
The influence and fate of heavy metals (Pb, Zn Cu, Cr, Ni and Cd) are determined during bacterial growth in a medium composed of used motor oil. Growth is apparently not affected by the relatively high level of metals found in the oil. The metals are transferred into the aqueous phase during bacterial growth. The relation between bacterial growth, hydrocarbon metabolization and metal solubilization is analyzed. In this paper, the concentration of cadmium in used motor oil is reported.
INTRODUCTION
The product ion of lubricating oil in the Western countries corresponds to 1--2% of the total amount of refined petroleum [1] . Combust ion losses and oil leaks from the engine during use may represent 35% of the oil consumed [2] . In 1980 more than 9 million tons of used motor oil was produced in the United States [2] , with only a small a m o u n t of this being re-refined, and some used as an auxiliary combust ible (3.32 and 40%, respectively, in the U.S.A.) [3] . Therefore, it appears that most used motor oil, several million tons, is dispersed to the environment each year.
Used motor oil is a very dangerous pollutant [7- -10] , and its toxici ty has been established in several reports [7, 9, 10, 14] . It contains carcino- genic substances such as polyaromatic hydrocarbons (PAH) (e.g. benzo[a] - pyrene). These compounds are much more important in used motor oil than in new oil [8, 9 ] , since most of the PAHs dissolved in petrol are not emit ted in the combust ion gas but accumulate in the oil [15] . For example, benzo- [a]pyrene has been found in used oil at a concentration of 2 2 p g g -1 [7] . Used motor oil also contains a high level of metals [2, 11--13] , so disposal by burning is not a good solution, because heavy metals are dispersed to the atmosphere during the process [4--6] .
Several studies have already been carried out on the biodegradation of lubricating oil [13, 16- -18] , and the present work was intended to
0048-9697/86/$03.50 © 1986 Elsevier Science Publishers B.V.
110
determine the effect of heavy metals on the biodegradation of used lubricating oil by bacteria and also to establish the fate of these metals during the process.
EXPERIMENTAL
The experiments were carried out at least in triplicate.
Microorgan isms
Pseudomonas fluorescens, strain B-52 of the Depar tment of Plant Biology of the University of Geneva.
Conditions of culture
Pseudomonas fluorescens were cultivated in a fe rmentor flask containing 11 of Turfreijer-modffied medium [19] : 15 g of lubricating oil (new, entire used or filtered used); 7.5 gNH 4 NO 3 ; 0 .3gMgSO 4 • 7H20 ; 0.3 gK2HPO4; 3 g Tween 80; and 0.0026 g iron citrate, added to 11 of distilled water.
The pH was automatically maintained at 7.2 with a solution of 0.5% NH4OH. The cultures were stirred magnetically at 20°C and were well aerated by air bubbling (1.221 min -1 ).
Protein estimation
According to Lowry [20] .
Motor oil
Commercial new lubricating moto r oil " E l f " 15W-40 was used. The used m o t o r oil was a mixture collected f rom several cars in a service station. The filtered used oil was obtained by fi l tration of used oil, dissolved in chloroform, on a Whatman filter 0.2/am WCN type and solvent evaporation under vacuum at 70°C.
Determination of heavy metals
Metals were determined with an atomic absorption spect rometer (AAS) with carbon furnace Perking-Elmer 2280. Samples were prepared from different fractions derived from moto r oil and bacteria cultures. All equip- ment was pre-soaked in 10% HNO 3 for 2 4 h and washed with double distilled water.
Motor oil. Motor oil ( l g ) (new, entire used or filtered used) was evaporated at 600°C and the residue was mineralized with 1 ml of HNO 3 (85%).
111
Used oil particles. The particles remaining on the filter after filtration of 1 g entire used oil were mineralized with 2 ml of HNO3 while heating.
Dissolved metals. Culture (30 ml) was centrifuged at 20 0 0 0 g for 15 min; the oil was removed as the upper layer and 20 ml of the aqueous phase was filtered (0.2 pm) and acidified with 1 ml of HNO 3 .
Pelleted bacteria and immobilized material. Bacteria and precipitated material were collected by centrifuging at 2 0 0 0 0 g for 15min. Bacteria were grown in a medium free of particles (filtered used oil). Metals, adsorbed to bacteria and precipitated, were determined non-specifically after com- plexing with EDTA. The bacteria pellet was washed in 30 ml of a mixture of EDTA/hexamethylente t raamine (1:3) at an EDTA concentrat ion about 50 times higher than the concentrat ion of metals. The pH was adjusted to 9.0 by the addition of NaOH. Complexat ion was completed after 30 min and the suspension was centrifuged at 20 0 0 0 g for 15 rain. The supernatant contained the previously adsorbed and precipitated metals. The bacteria pellet was mineralized with 2 ml of HNO3 and contained the assimilated metals.
Biodegradation of used motor oil
Samples of the culture medium were taken periodically during bacterial growth and hydrocarbons were extracted several times with chloroform after acidification with H 3 PO4. The solvent was evaporated under vacuum (50°C) and total hydrocarbons were determined by gravimetric analysis. The residue weights of the oil were normalized for volatilization losses of hydrocarbons during evaporation of solvents. The fa t ty acids were separated from total hydrocarbons and weighed according to McCarty and Duthie [21] .
Gas--liquid chromatography
The fa t ty acids fraction was diluted with dimethylformamide (DMF) and methyla ted with methanol /methyl iodide/ te t ramethylammonium hydroxide. This mixture was diluted with water and subsequently washed with n-hexane and evaporated under reduced pressure. Chromatography of the methyla ted fat ty acids was performed on a Finnigan 9610 gas chromatograph equipped with a 2 0 m × 0.32 mm column, type WCOT, SE54 and a hydrocarbon flame-ionization detector. The oven temperature was held for 1.2 min at 60°C after sample injection (splitless) and was subsequently raised to 150°C at a rate of 10°C min -1 , and then to 250°C at a rate of 4°C min -1 , where it remained for 30 min.
RESULTS
Analysis of the lubricating oil
The metal content of new and used oils, determined by AAS, is shown
112
in Table 1. Our values are consistent with those obtained by Cotton et al. [11] on 30 samples of different used oils (see Table 1). Information on the presence of cadmium in used lubricating oil is lacking, as already pointed out by Broeker and Gliwa [4], and the only values we can refer to are those reported by Lagwerff and Specht [22] on new lubricating oil. Their values are consistent with ours, and we assume, therefore, that the amount of cadmium determined in used oil is correct.
Almost all metals are in lower concentrations in the new motor oil than in the used oil, except for zinc. We must remember that zinc is added to the oil as a detergent, such as zinc dialkyl- or diaryl<iithiophosphates, and as other lubricating oil additives [1, 23, 24] , ifi proportions dependent on the production company. In the used oil almost all of the zinc and the copper, but only half of the cadmium and one-third of the other metals are dissolved or in the form of small particles (< 0.2 pm).
The used oil analyzed in this work came from engines which had been supplied with lead-containing petrol. If lead-free petrol had been used, the used oil would have contained less lead but the same amount of the other metals, as was found for oils of diesel engines [13].
Grow th o f Pseudomonas fluorescens
The influence of metals on bacterial growth was tested by using three different culture mediums, prepared with: (a) new motor oil, (b) entire used oil; and (c) filtered used oil (0.2 pm) from which an important amount of metals had been eliminated (see Table 1).
Bacterial growth is apparently indentical in the three different mediums (Fig. 1). It should be mentioned that sometimes, when using different used oils, differences in growth have been noted. This phenomenon may be related to heterogeneity in the hydrocarbon composition, as no relation has been found between the bacterial growth and the metal content of the oil.
The non-inhibitory effect of metals could be attributed to the fact that they are in a non-accessible form. To assess clearly any inhibition of bacterial growth by metals, two culture mediums were prepared, one with 0.2% ammonium lactate and the other with 1.5% new motor oil, and various amounts of soluble metals (concentration in/~gg-1 Pb, 50; Zn, 20; Cu, 0.3; Cr, 0.015; Ni, 0.009; and Cd, 0.008), in order to obtain approximately the concentration found in the culture medium prepared with used motor oil. No inhibition could be detected in the concentration range used. Petrol (9% w/w in oil) and particles (200pgg - l ) in the medium were not inhibitory.
Cessation of bacterial growth is due to the exhaustion of metabolizable hydrocarbons, as is confirmed by P. fluorescens growing on the mediums containing different proportions of mineral components and oil (not shown). Pseudomonas fluorescens cannot grow in a medium prepared with bio- degraded oil, and the dilution of the medium during growth shows that this is not due to the accumulation of inhibitor compounds.
TA
BL
E
1
CO
NC
EN
TR
AT
ION
O
F M
ET
AL
S
IN N
EW
MO
TO
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OIL
AN
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NT
IRE
A
ND
F
RA
CT
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S
OF
US
ED
LU
BR
ICA
TIN
G
MO
TO
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OIL
Met
al
Con
cen
trat
ion
of
met
als
(pg
g-l
) in
New
m
otor
oi
l
Pb
0.48
0 Z
n
1482
.00
cu
0.20
4 N
i N
Dd
Cr
ND
C
d 0.
087
En
tire
use
d m
otor
oi
l
3091
.50
(1O
O)h
11
76.0
0 (3
5.99
) 35
8500
(6
3.78
) (0
.23)
70
79.0
13
885
1128
.50
(100
) 89
6.00
(7
5.12
) 40
675
(19.
82)
(5.0
6)
1061
.0
2500
6.
04
(100
) 6.
77
(106
.14)
55
(5
.06)
(1
1.20
)=
28.0
56
0.
556
(100
) 0.
223
(37.
94)
96 (
94.9
6)
( 32.
90)c
1.
2 5
1.00
0 (1
00)
0.30
0 (2
8.34
) 34
(1
8.70
) (5
2.96
) 10
.5
24
0.47
9 (1
00)
0.23
8 (4
9.69
) 32
(3
6.74
) (1
3.57
) N
D
ND
Use
d m
otor
oi
l fr
acti
onsa
Fil
tere
d u
sed
mot
or
oil
Par
ticl
es
Vol
atil
es
and
loss
Use
d m
otor
oi
l ac
cord
ing
to
Cot
ton
et
al.
[ 111
Ave
rage
M
ax.
‘Du
rin
g u
sed
oil
frac
tion
atio
n,
96.6
1%
of
tota
l m
ass
was
rec
over
ed
in
the
filt
ered
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0.55
%
in t
he
part
icle
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84%
in
th
e vo
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and
loss
. bT
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n p
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ndi
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tage
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th
e va
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s fr
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ons.
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min
atio
n.
ND
= N
ot d
eter
min
ed.
114
25C
E
"{ 200
.c 15C
0
"5 100 ID
5O
Time (days)
Fig. 1. Bacterial growth in: (©) 1.5% new m o t o r oil; ($) 1.5% entire used mo to r oil; and (v) 1.5% fi l tered used m o t o r oil.
On a weight basis, the hydrocarbon conten t of the medium decreased by 17.08%, while the fraction of oxygenated hydrocarbons, such as fa t ty acids, increased f rom 3.64 to almost 12.0% (Table 2). The appearance of long- chain f a t ty acids detected by GLC is indicative of an oxidative process (Fig. 2).
The growth values in entire used oil (Table 2) fits the Gomper tz equation:
1 dW _ pe-st
W d t
where W 0 = 0.42, p = 11.898, and S = 1.897, and the metabolizat ion (hydrocarbon loss) can be related to a threshold response curve:
a t n M -
b" + t"
where a = 3.165; b = 2.924; n = 1.5; W represents the amount of protein in micrograms per milliliter of culture, M is the hydrocarbon losses in grams; t is the time in days; and p, s, a and b are constants.
The growth rate (dW/dt) and the metabolizat ion rate (dM/dt) reach a maximal value more .or less at the same time, but the metabolizat ion rate increases before the growth rate, and decays less rapidly (Fig. 3).
Behaviour of metals during growth
The fate of metals in used motor oil was determined during bacterial growth. Samples were taken at different stages of culture, and the level of
100-
115
RIC
100.
RIC
q j.
~b ~o 50 4o so Time (min)
Fig. 2. GLC chromatograms of fatty acid (methyl esters) fraction (a) before bacterial growth and (b) after 8 days of bacterial growth.
150 .~
~ a6
! t/ 1 ,o t '
o o 1 2 3 4 5 6 7 8 9
Time (days)
Fig. 3. Metabolization and growth rates relation. (o) Growth rate (dW/dt) and (o) metabolization rate (dM/dt).
meta l d isso lved in the a q u e o u s phase was d e t e r m i n e d by A A S . A transfer o f t h e meta l s in to t h e a q u e o u s phase is observed (Fig. 4) , w i t h the e x c e p t i o n o f lead. This proces s s l ows d o w n w h e n g r o w t h s tops , and enters the s ta t ionary phase .
116
TABLE 2
BACTERIAL GROWTH AND BIODEGRADATION IN USED MOTOR OIL
Time Growth Hydrocarbon loss a
(days) (pg protein m1-1 ) (g1-1 ) (%)
% of fatty acids
in residual oil
0 0 0 0 3.64 1 93 (+ 24) b ND c ND ND 2 185 (+- 43) 1.152 (+ 0.12) 7.68 6.84 3 218 (+ 38) ND ND ND 4 215 (+ 15) 1.932 (-+ 0.17) 12.88 9.96 5 223 (+3) ND ND ND 6 235 (+ 16) 2.391 (+ 0.20) 15.94 11.64 7 221 (+ 19) ND ND ND 8 239 (-+ 13) 2.562 (+ 0.29) 17.08 11.97
aThe initial hydrocarbon content in 11 of 1.5% used motor oil medium was 1.5 g. Values are normalized for volatilization losses of hydrocarbon during extraction procedure. b Standard deviation is derived from three replicate experiments. CND = not determined.
The same behavior was observed in all exper imen t s , in spite o f d i f ferences in the initial c oncen t r a t i on of metals in the m o t o r oil. It is n o t re la ted to change in the [H +] concen t r a t i on , as the pH was main ta ined at 7.2 th rough- o u t the expe r imen t . No significant t ransfe r could be de t ec t ed in the con t ro l w i t h o u t bacter ia . I t can, the re fore , be deduced tha t this t ransfer is l inked to bacter ia l g rowth . The ne t t ransfer of metals in to the aqueous phase af te r 8 days o f bacter ial g rowth (Table 3) demons t r a t e s tha t all metals , e x c e p t lead, are t rans fe r red f r om the oil to the aqueous phase. A lmos t half o f the zinc and the coppe r and one-quar te r o f the cadmium is t ransfer red in to the aqueous phase by bacter ial g rowth af te r 8 days. The lead c o n t e n t is on ly slightly lowered .
I t was n o t possible to de t e rmine the concen t r a t i ons o f dissolved c h r o m i u m and nickel, as t hey were below our de t ec t i on limits (0 .002 pg g- i fo r Cr and 0 . 0 0 4 p g g -1 for Ni).
A f u r t h e r e x p e r i m e n t was carr ied o u t to dist inguish be tween the adso rbed /p rec ip i t a t ed and the assimilated metals in a m e d i u m conta in ing 1.5% of f i l tered used m o t o r oil (free o f particles). Af te r 8 days o f growth , bacter ia were pe l le ted by cen t r i fuga t ion and the pel le t was washed wi th E D T A / h e x a m e t h y l e n t e t r a a m i n e . The suspension was cen t r i fuged , and the previous ly adsorbed and p rec ip i t a ted metals were recovered in the super- na tan t . The bacter ia lJellet con ta ined the assimilated metals .
As shown in Table 4, heavy metals are assimilated by P. fluorescens ( c o n c e n t r a t i o n in # g g - ' d ry weight of bacteria: Pb, 148; Zn, 144, Cu, 27; Ni, 0.98; Cr, 0 .574; and Cd, 0 .221) . I t can be deduced tha t there is ef fec t ive b ioaccumula t i on and tha t the relat ively high concen t r a t i ons o f heavy metals do n o t r ep re sen t a tox ic level (Fig. 1). I t also appears tha t these
117
4000
2000
400
~, 2ooc
o
~ 0 E
~ 60 a
4C
20
1,5
1,0
0,5
Pb
i , t
J , i
Time (days).
Fig. 4. Dissolved heavy metals in aqueous phase during bacterial growth.
microorganisms have the capacity of adsorbing and precipitating metals, especially Ni and Cd. Therefore, immobilization of metals by bacteria is efficient through assimilation, adsorption and precipitation. After 8 days P. fluorescens growth, 10--35% of metals contained in oil are immobilized.
DISCUSSION
The strain of P. fluorescens used in this work is fully able to grow on a medium supplied with mineral oil as an organic source. The high level of heavy metals in used lubricating oil does not affect growth. These metals, known for their toxicity, are apparently without effect on the bacteria (Fig. 1).
A portion of the hydrocarbons are mineralized and a wide range of
118
TABLE 3 BACTERIAL TRANSFER OF METALS INTO THE AQUEOUS PHASE a
Metal Concentration (pg l -I )
Total Initial in In aqueous phase in medium aqueous phase after growth
Net bacterial transfer into aqueous phase
Pb 46372.50 (100) b 4075.00 (8.79) 4000.00 (8.62) -75.00 (-0.16) Zn 16927.50 (100) 1950.00 (11.52) 8850.00 (52.28) 6900.00 (40.76) Cu 90.60 (100) 13.10 (14.46) 64.00 (70.64) 50.90 (56.18) Cd 7.18(100) 0.63 (8.77) 2.41 (33.56) 1.78 (24.79)
aBacteria were grown in the 1.5% entire used motor oil medium for 8 days. bThe numbers in parentheses indicate the percentage of metals.
long-chain fat ty acids are produced by the bacteria (Table 2 and Fig. 2). The fa t ty acid production is indicative of a normal metabolic pathway for microbial oxidations of hydrocarbons.
A large amount of metals was recovered in the aqueous phase and a smaller port ion was immobilized by precipitation, adsorption and assimilation (Tables 3 and 4). The transfer into the aqueous phase can be the consequence of metabolization of hydrocarbons liberating the metals previously bound or complexed. After filtration of used oil, a large amount of metals remains in the filtrate (see Table 1). These metals are either bound or complexed to hydrocarbons or present as small elemental particles (< 0.2 #m). The negatively charged electron cloud of hydrocarbons can fix positive ions and form metal complexes [25]. The hydrocarbon capacity of com- plexing metals has been illustrated by Walker and Colwell [26], who measured the concentration of metals in a sea bay and found that the concentration in oil was much higher than that in the sediments and in the water, and that the percentage of several metals (Zn, Cr, Pb, Cu, Ni, Cd and Hg) was proportional to the oil content of the sediments.
A portion of the transferred metals may come from the elemental particles. Pseudomonas fluorescens are known to transform elemental metals by oxidation [27]. Another possible cause of metal transfer into the aqueous phase, may be the production by P. fluorescens of chelating agents which solubilize these metals. Bacteria in general [28, 29] , and Pseudomonas sp. in particular [30], are able to produce extracellular polysaccharides and other organic substances which emulsify hydrocarbons.
The values of metal adsorption and metal assimilation by P. fluorescens obtained in this work (Table 3) confirm the high capacity for adsorbing and accumulating metals by this microorganism [27, 31].
Adsorption is a simple non-specific binding of metals to cell surfaces, slime layers, extracellular matrices, etc. It is well known that metals bind
TABLE 4 IMMOBILIZATION OF METALS BY ADSORPTION AND PRECIPITATION a
119
BACTERIA THROUGH ASSIMILATION,
Metal Amount of metals in 11 of growth medium (pg)
Initial Assimilated Adsorbed and Total in medium by bacteria b precipitated immobilized
Pb 17640.00(100) b 61.92 (0.35) 2232.22 (12.65) 2294.14 (13.00) Zn 13440.00 (100) 60.25 (0.45) 1203.35 (8.95) 1263.60 (9.40) Cu 101.55(100) 11.30 (11.12) 7.95 (7.83) 19.25 (18.95) Ni 3.34 (100) 0.410 (12.27) 0.760 (22.73) 1.170 (35.02) Cr 4.50 (100) 0.240 (5.33) 0.524 (11.65) 0.764 (16.98) Cd 3.57 (100) 0.092 (2.59) 0.854 (23.92) 0.946 (26.51)
aThe bacteria were grown in the 1.5% filtered used motor oil medium for 8 days. bAfter 8 days growth, the bacterial concentration in dry weight was 418.41 mg 1-1. CThe numbers in parentheses indicate the percentage of metals recovered in each fraction.
strongly to cell surfaces, specifically to the proteins and lipids such as polygalacturonic acid, an important compound in the consti tut ion of exterior layers of bacteria cell [32] . The adsorption can lead to metal precipitation when, after several cellular divisions, the bacteria are liberated from the mucilage layer, leaving a solid insoluble body containing the metals [33] . Bacterial extraceUular polymers may also complex metals [34] .
Finally, metals can be immobilized by intracellular assimilation, in an accumulat ion pool where they are complexed with sulfhydryl compounds, in order to alleviate their toxic effects [32] .
By comparing the behavior of metals (Fig. 4) with the growth rate and the metabolizat ion rate (Fig. 3), one can see that precipitation and some transfer to the aqueous phase are coincident with the increase of growth rate. When growth rate decreases, transfer to the aqueous phase is increased, especially when the difference between metabolization rate and growth rate is accentuated. This can be explained as follows: metals are liberated from the oil phase by metabolizat ion and a proport ion of the metals found in the aqueous phase are immobilized (adsorption, precipitation and assimilation) during bacterial growth. The observed solubilization of metals is, therefore, dependent on the balance between growth and metabolization.
In summary, our results show that the high content of heavy metals in used moto r oil has no inhibitory effect on the growth of P. fluorescens. This bacteria is capable of assimilating and adsorbing a large quant i ty of heavy metals.
A transfer of metals from the oil phase to the aqueous phase is observed during growth, when the metabolization rate exceeds the growth rate. This is of importance in relation to the environment, because the transfer involves the increase of heavy metal mobil i ty and, in consequence, their toxicity. The disposal of used lubricating off by bacterial biodegradation has, therefore, to be considered with caution.
120
ACKNOWLEDGEMENTS
We thank Mr A. Balikungeri of the Department of Inorganic, Analytical and Applied Chemistry of the University of Geneva for his greatly appreciated assistance. We also thank Mr P. Simon for critical reading of the manuscript.
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