Bacterial degradation of crude oil

most unlikely that similar circumstances will occur again. It is, however, unfortunately true that it is only a question of time before another massive spillage occurs as a result of collision or some other disaster in the heavily congested waters around the British Isles. National and local plans now exist at most levels to meet such a situation. Local organizations are kept busy by the continual minor spillages that still occur, particularly around the busier shipping lanes. The same incidents serve to test the many lines of communications between the various authorities that are now actively involved in combating this problem.

The Royal Navy is closely involved in the national arrangements for dealing with oil pollution and is ready to respond to any threat that may arise. It also hopes that the need for its services will decrease as international agreement reduces the likelihood, and level, of oil pollution at sea. Office of the Flag Officer, Plymouth, Mount Wise, Devonport.

J. A. F. Lawson

Bacterial Degradation of Crude Oil Since November 1968 we have been concerned with (1) identifying the bacteria involved in the breakdown of oil and the optimum conditions for their growth; (2) observing the changes which occur in the bacterial populations during the course of oil degradation; (3) observing the changes induced by the bacteria in the composition of the oil and the time constants of these changes. Bacterial strains able to utilize crude oil or single hydrocarbons as sources of carbon and energy were isolated from estuarine mud after enrichment in a seawater medium containing crude oil (Kuwait, supplied by BP, Llandarcy), or a single hydrocarbon, as the only source of carbon. Octane, pentadecane and hexadecane were used as examples of straight chain paraffins with odd and even C-numbers; 2 methyl-hexane was used as an example of an isoparaffin and cyclohexane as an example of a cyclo-paraffin; heptene as an olefin; 1 phenyl-dodecane as a phenylalkane; and benzene and naphthalene were used as examples of aromatic hydrocarbons. TABLE 1 Growth of Isolates in Various Hydrocarbons

Approximately fifty strains of bacteria were collected and subjected to further examination. The majority were

Pseudomonas, Acinetobacter, Achromobacter/Alcaligenes, Flavobacterium and a few Gram positive organisms which are so far unidentified. Their ability to utilize various hydrocarbons as the sole source of carbon and energy is shown in Table 1. At present the action of these isolates, and also of mixed cultures, on mixtures of hydrocarbons is being determined. The results of gas-liquid-chromatography (GLC) indicate that in a mixture of C12 Cl s and C16 alkanes, the Cl2 is always attacked first and afterwards there is no preference between C1 s and C 16Growth of some of these strains and also of mixed cultures is not markedly inhibited in the presence of the following sinkers: siliconized pulverized fly ash, siliconized sand, Grangemouth 1 and 2 (BP), spent tannery lime, Crowsink, Oilsink, stearated chalk whiting, Nautex-H, untreated fly ash, and sand. In some tests bacteria in both pure and mixed culture were dried on to sinkers at 37 deg. C. The viability of the organisms was tested and was found to vary with different strains of bacteria as well as with different types of sinkers.

Growth o n s u n k e n oil Attempts to measure bacterial growth on oil sunk with any of the sinkers already mentioned were accompanied by practical difficulties. It was necessary to extract the bacteria from the oil and to ensure even distribution of the sample for counting. The most practical way was to emulsify the sample. Several agents were tested including Polycomplex-A, Nonidet P-40, Tween 80 and four other compounds; Corexit (Esso) was found to be the most suitable. Kuwait and Nigerian crude oils were mixed with siliconized fly ash and inoculated with a mixed culture of bacteria. Population changes were followed by counting the number of bacteria present in the inner and outer layers of oil (Fig. 1). The number of bacteria increased on both types of crude oil, and one organism eventually became dominant. The outer layer o f oil supported a greater population than the inner layer where it is assumed a deficiency of oxygen inhibited growth. A further experiment where oxygen was excluded as far as possible has verified this result. GLC analyses of the residual Nigerian and Kuwait crude oil are n o t complete and the results of this work will be given later. The results obtained so far, however, indicate that most n-alkanes have been almost completely metabolized in 2 months at 30 deg. C whereas the iso-alkanes 25

FOTAL NUMBERS of BACTERIA m NIGERIAN CRUDE OIL TOTAL NUMBERS of BACTERIA on KUWAIT CRUDE OIL

~2-

12o

o 11-

o Outer layer

•o Outer layer & A

t~

70-

Irloer layer

9.

o

o c~

E

E

}s-

:2 5

i

[ ,b

2'0

3b

~me of mcubation at

zb 30"C (days)

5b ;,"e

f 'no ~bat/on at

30"C

(days)

Fig. ia.

~Yig. lb.

have not been attacked. There is no observable difference in the remaining mass of oil.

populations and activity. Changes in the compositions of the oil will also be studied. Now that stocks of crude oils and w e a t h e r e d crude oils are readily available, the w o r k will be e x t e n d e d to cover investigation of the microbial degradation of m o r e types of crude oil. We thank Professor D. E. Hughes for his help and advice and Miss Susan J e n k i n s for technical assistance. This w o r k was supported by a grant from the Ministry of Technology,

Future w o r k

Because growth m e t h o d s for examining microbial utilization of hydrocarbons have not b e e n entirely satis-~ factory, we hope to measure activity using respirometric techniques in order to gain more e x a c t information. Measurement of o x y g e n uptake using an oxygen electrode was unsatisfactory, and it is likely that the m a n o m e t r i c methods previously used by G. J o n e s in this d e p a r t m e n t will be more useful. The w o r k on the sequence of attack of mixtures of hydrocarbons will be extended. We hope to carry out experiments in natural conditions using sunken oil, examining the changes with time in bacterial

Testing for Toxicity When a toxicity test is required before effluents can be discharged into British rivers the rainbow trout should be used, in a modification of the m e t h o d developed by the Trent River Authority. A c o m m i t t e e of experts has recomm e n d e d to Mr A n t h o n y Greenwood, Minister of Housing and Local G o v e r n m e n t , that the toxicity of an effluent may be defined as the n u m b e r of volumes to which one volume of a sample must be diluted so that five out of ten rainbow trout survive for 48h at 15 deg. C in a specified solution of standard water ('Fish T o x i c i t y Tests', HMSO, 2s 6d). The c o m m i t t e e r e c o m m e n d e d this test in preference to that developed by the Ministry of Agriculture, Fisheries and Food, which differs principally in requiring that the test solution be continually replaced to provide an adequate 26

D e p a r t m e n t of Microbiology, University College of S. Wales and Monmouthshire, Cathays Park, Cardiff, DF1 3NR.

J u n e A. Byrom Sally Beastall Sylvia Scotland

supply of oxygen. The r e c o m m e n d e d test, however, requires only that air be bubbled through the solution. After comparing measurements o b t a i n e d by b o t h methods, the c o m m i t t e e decided that a standardized version of the test developed by the Trent River A u t h o r i t y will be adequate for control purposes. River authorities, sewage boards, and local authorities made considerable progress in the control of pollution w i t h o u t a fish toxicity test since the Rivers (Prevention of Pollution) Act in 1951, which required testing or analysis of sewage and effluents. In m a n y cases w h e n the nature of the effluent is known toxicity can be measured by chemical analysis. But w h e n no such analysis is possible, if for example the c o m p l e x constituents of a discharge are not fully known, it is necessary to use a test involving fish.