- Email: [email protected]
Treatment of petroleum industry oil sludge in soil
T I B T E C H - A U G U S T 1986
References 1 Jensen, F. K., Blythman, H.E., Carriere, D., Casellas, P., Gros, O., Laurant, J.E., Paolucci, F., Pau, B., Poncelet, P., Richer, G., Vidal, H. and Voisin, G. A. (1982) Immuno]. Rev. 62, 185-216 2 Pirker, R., FitzerGerald, D.J., Hamilton, T.C., Ozols, R.F., Laird, W., Frankel, A. E., Willingham, M. C. and Pastan, I. (1985) Jo Clin. Invest. 76, 1261-1267
3 Vitteta, E. S. and Uhr, J.W. (1985) Annu. Rev. Immunol. 3, 197-212 4 Dorland's Illustrated Medical Diction-
ary (1974) (25th edn), p. 767, W.B. Saunders Company 5 Stedman'~s Medical Dictionary Illustrated (1979), p. 694, Williams and
Wilkins 60lsnes, S. and Phil, A. (1982) J. Pharmacol. Ther. 15, 355-381 7 Youle, R. J. and Neville, D. M. (1980) Proc. Natl Acad. Sci. USA 77,
Treatment of petroleum industry oil sludge in soil K. Shailubhai
5483-5486 8 Volkman, D. J., Ahmad, A., Fauci, A. S. and Neville, D. M. (1982) J. Exp. Med. 156, 634-639 ATEEQ AHMAD
Division o f Biochemistry, Departm e n t o f the A r m y , Walter Reed A r m y Institute of Research, Washington, DC 20307, USA.
accumulate these hydrocarbons in their fatty tissues and hence are able to transfer the toxic compounds to higher trophic levels, including humans. Therefore, the concern over chronic hydrocarbon inputs is of importance to many coastal communities. The ecologically acceptable disposal of oil sludges and other waste hydrocarbons is a major challenge confronting the petroleum industry.
Biodegradation ofoil sludge in soil Petroleum refining unavoidably generates large volumes of oil sludge. The environmentally acceptable disposal of oil sludge is a current challenge to the petroleum industry. Many soil microorganisms possess a remarkable capacity to degrade various components of crude oil. The land treatment of oil sludge - land farming - is an environmentally acceptable and economically feasible disposal method. The development of efficient hydrocarbon-degrading microorganisms and their use for cleaning oil sludge in soil are discussed. Petroleum refineries use water in many operations during the refining and cracking of heavier hydrocarbons. In the process the water gets contaminated with various chemicals. Therefore, the chemical content of petroleum refinery oil sludge consists of not only the compounds from the original crude oil stock but also those produced in fracturing processes and chemical additives used within the refining operations. The major components of oil waste include metallic and non-metallic compounds and water. The principal metallic constituents of refinery waste are zinc, chromium, vanadium, nickel, lead and copper. The main non-metallic constituents are K. Shailubhai is at the Department of Animal Sciences, University of Mary]and, College Park, MD 20742, USA.
organics such as n-alkanes, paraffins, olefins, aromatics, asphaltics, phenols and polynuclear aromatic hydrocarbons. The composition of oil sludge varies from batch to batch depending on the history of treatment and storage. Recent studies have shown that petroleum hydrocarbons can have both lethal and sublethal effects on a wide variety of marine organisms. The sublethal effects of petroleum hydrocarbons include interference with the chemotactic response of bacteria. This results in: inhibition of organic matter decomposition in sea water1; reduced photosynthetic rate in algae2; abnormal development and hatching of barnacle embryos3; and inhibition of moulting, alteration of sex ratios, and reduced resistance to environmental stress in c r a b s 4. Many fish have been shown to
(~ 1986, Elsevier Science Publishers B,V., Amsterdam 0166- 9430/86/$02.00
Soil provides a natural environment for the biodegradation of waste materials through complex physical, chemical and microbiological processes. Microorganisms present naturally in soil degrade the waste oil and produce intermediate products such as alcohols, phenols, esters, aldehydes, ketones and fatty acids. These products are ultimately converted to carbon dioxide, water and cellular materials. Although microbial assimilation is the principal means of waste degradation, the contribution of other non-biological processes (chemical and photochemical autooxidation, evaporation and volatilization) is also significant. The overall rate of biodegradation is influenced by the type of oil sludge, by the microorganisms present in soil and by the climate. The ability of microorganisms to degrade various components of petroleum has been recognized for more than a century. More than 100 species of bacteria, yeast, actinomycetes and fungi, representing 31 genera are known to attack one or more types of petroleum hydrocarbons 5. Atlas 6 listed 22 genera of bacteria and 14 genera of fungi in which hydrocarbon degradation ability had been demonstrated. Recent
TIBTECH- AUGUST1986
Table 1 Microbial genera degrading hydrocarbons in soil
Bacteria Achromobacter Aerobacillus Alcaligenes Arthrobacter Bacillus Bacterium Beijerinckia Botrytis Citrobacter Clostridium Corynebacterium Desulfovibrio Enterobacter
Escherichia Flavobacterium Gaffkya Methanobacterium Micrococcus Micromonospora Mycobacterium Pseudomonas Sarcina Serratia Spirillum Thiobacillus
studies continue to expand the list of microbial species that have been shown to be capable of degrading various components of petroleum 7'8. Table 1 lists the primary microorganisms responsible for hydrocarbon degradation in soil. The ubiquitous distribution of yeasts, fungi and bacteria in oilcontaminated environments has led to the conclusion that microorganisms play an important role in the degradation of oil in soil. Llanos and Kjoller (1976) found that the application of waste oil to soil favored the growth of organisms capable of utilizing crude oil as sole sources of carbon and energy 9. Microbial distribution patterns in a typical soil indicate that the upper soil zone has by far the largest microbial population and, thus, may be the most active zone for waste biodegradation 1°. The application of oil waste to soil causes the enrichment of specific hydrocarbon-degrading microorganisms. Raymond et aL reported an increase in the soil microbial population due to oil application from 1 x 105 to 1 x 1 0 7 microorganisms per gram of soil. 1~
Oil sludge disposal technology Oily sludges are derived from o i l water separators, tank bottoms or cleanings, air flotation treatment of waste waters, and cleanings from lagoons or oxidation ponds. A major consideration in treating oily sludges is that these materials are generated from various sources and are discharged at irregular intervals. Thus, sludge composition is highly vari-
Actinomycetes
Fungi
Yeasts
Actinomyces Endomyces Nocardia
Aspergillus Cephalosporium Cunninghamella
Candida Rhodotorula Torula Torulopsis Trichoderma Saccharomyces
able. The most common processes for handling oily sludges are gravity thickening, vacuum filtration or centrifugal dewatering, and ultimate disposal is by dumping into the oceans, into lagoons, incineration, or land farming (landfill) (Fig. 1). However, dumping into the ocean is not recommended because aquatic organisms can rapidly accumulate heavy metals and toxic organic substances. Incineration is also no longer considered to be desirable because of the energy cost and the air pollution problems that are often associated with it. The disposal of oily sludges on soil is acceptable if it can be shown that such disposal will not contaminate ground water and will not create a seepage problem. Land farming operations that use soil microorganisms to degrade and clean up the oil satisfy these requirements.
Land farming of oily sludges has been successful at refineries where sufficient area of land is available for proper decomposition of oily sludge. The technique involves spreading the oily sludge in 10-15 cm layers, allowing the sludge to dry for about one week, adding fertilizers and then discing the sludge into the soil. Most major oil refineries in the US dispose of at least a portion of their oily wastes by land farming. Land treatment is also practised in the UK, the Netherlands, Sweden, Denmark, France and New Zealand 12.
Factors affectingbiodegradation During the treatment of oil waste, the microorganisms use hydrocarbons as sources of food and energy which results in the clean-up of oil waste effluent.
Fig. 1
Oil--water separation
Dewatering
Final disposal ~,-[ Incineration
Gravity
Filtration
~[ Landfarming ~[
Oil sludge disposal technology
Lagoon Ocean disposal
T I B T E C H - A U G U S T 1986
Composition of waste The degree and rate of microbial biodegradation depends on the nature and type of hydrocarbons present in the oil waste. Among the various petroleum hydrocarbons, the n-alkanes of intermediate chain length are preferred substrates for microbial degradation, whereas highly branched ~ a l k a n e s and cyclealkanes in particular are less susceptible to microbial attack 13. Complex alicyclic compounds such as hopanes are among the most persistent components of petroleum waste 7. Aromatic hydrocarbons are also abundant in oil waste and most of them are biodegradable. However, some of them, particularly polynuclear hydrocarbons, are metabolically recalcitrant such that microbial treatment has very little effect on their fate. In addition, the viscosity, solubility and toxicity of waste constituents also exert a significant influence on their degradability. Two phenomena which need to be considered in the biodegradation process of oil sludge are 'sparing' and 'co-oxidation'. When a microorganism with a broad substrate range is offered more than one type of organic substrate, it is likely that it will not attack the substrates simultaneously, but in a definitive sequence. For example, the degradation of pristane was inhibited in the presence of the easily metabolizable substrate hexadecane ~4. The susceptibility of the various fractions of crude oil to microbial degradation is in the following order: saturate > aromatic > asphaltic fraction. Hence, during the biodegradation of oil waste, aromatic and asphaltic components are not degraded until the easily metabolizable saturated n-alkanes :are exhausted. The phenomenon of co-oxidation has also been shown to play an important role in the biodegradation of crude oil. The compounds which otherwise would not be degraded can be enzymatically attacked within petroleum mixtures because individual organisms can grow on some of the hydrocarbons within crude oil 14. Climateandnutrition Several climatic and nutritional factors also synergistically affect the
rate at which oil sludge is degraded in soil. The main factors are soil oxygen content, nutrients, moisture content, pH and temperature. The biodegradation of oil waste in soil is carried out mainly by aerobic microorganisms present in the upper layer of soil: therefore oxygen must diffuse into the soil pore space to be accessible to the microorganisms. Anaerobic microoganisms will also degrade oil sludge in an anaerobic atmosphere, with nitrates, nitrites and sulfates replacing oxygen as oxidants. The most rapid biodegradation rates occur in the presence of oxygen; to achieve maximum waste soil interaction and to provide sufficient air (oxygen), the soil is tilled frequently. Biodegradation of hydrocarbons requires the presence of nitrogen, phosphorus and potassium, as well as lower levels of zinc, calcium, manganese, magnesium, iron and sulfur; the latter elements are essential for microbial metabolism. The nutrients can be provided by application of fertilizers to the soil and this is necessary to maintain or enhance the biodegradation rate. The amount of oil waste degraded was found to double with the addition of fertilizers 1~. Of the minerals, iron has been considered as potentially limiting for petroleum biodegradation even in sea water containing nitrogen and phosphorus 16. Extremes of very wet or very dry soil markedly reduce waste biodegradation rates. Saturated soil may affect oil degradation due to low supply of oxygen whereas very dry conditions may affect the degradation rates due to insufficient moisture contents 16. Temperature and pH are other factors which affect the biodegradation rates significantly. For example, very acidic or very alkaline soil pH will retard the microbial growth and activity. Temperature also affects the abiotic removal of oil waste from soil.
Enhanced oil sludge degradation The preceding discussion suggests that the success of oil sludge biodegradation by land farming is influenced by a complex mix of chemical, physical and biological factors. Though oil sludge biodegradation by land farming has been
successful, important parameters, such as the effect of tillage, soil texture and fluctuation in temperature during a 24 hour cycle have not been studied, Moroever, the oil sludge biodegradation in soil is a very slow process. The limitations, side effects, and high expense of traditional clean-up technology has stimulated interest in unconventional alternatives, such as the use of hydrocarbon-degrading microorganisms for clean-up or increasing the biodegradation rate artificially by providing optimum conditions. It appears possible to enhance oil sludge biodegradation, in an enclosed, temperature controlled aerobic system by inoculation of highly efficient oil degrading microorganisms, along with some mineral nutrients. Unlike 'land farming', this method of oil sludge disposal is less dependent on favorable weather conditions and is equally amenable to manipulation. This approach has been used successfully to develop a treatment method for the biodegradation of oil sludge: Rhodotorula rubra, a yeast strain found to be capable of degrading a wide range of aliphatic and aromatic hydrocarbons, was inoculated in oil sludge along with some mineral salts in a temperature controlled (28°C) stainless steel fermentor 17'18. Aeration was provided by compressed filter-sterilized air. Under similar conditions uninoculated controls and controls to which azide was added to prevent microbial activity were also incubated. Samples were removed for analysis after seven days. As a result of biodegradation, there was 50-60% decrease in the chemical oxygen demand (COD) and 80-90% decrease in the biological oxygen demand (BOD) (Fig. 2). (The decrease in COD and BOD values can be taken as criteria for the degradation of hydrocarbons present in oil sludge.) There was also a 40% decrease in BOD and 14 OYodecrease in COD in the uninoculated control. This may reflect the activities of the microflora within the oil sludge itself. There was also some decrease in BOD (4.7%) and COD (8.0%) values in the azideadded control, which was attributed to the weathering of the volatile components of crude oil. At the end
T I B T E C H - A U G U S T 1986
--
Fig. 2
×10 0
~ 1
3 Time (days)
~ ~3~_
dation rate in land farming is very slow and is dependent on biological and climatic factors. These limitations can be overcome by mixing highly efficient hydrocarbon-degrading microorganisms in soil and by providing optimum conditions for biodegradation of crude oil. For instance, it has been shown that, by using Rhodotorula rubra, a very efficient hydrocarbon degrading yeast, the oil sludge under laboratory conditions could be degraded to an acceptable level within seven days of treatment. However, these findings will need validation and possible adjustments on the industrial scale. Nevertheless, it may greatly reduce and simplify oil sludge treatment on a larger scale. Future developments on the oil sludge treatment process will require a close collaboration between researchers in microbiology and bioengineering technology to implement full scale processes. Another aspect which requires further work is the development of microorganisms which can degrade various fractions of oil sludge completely in order to bring down its COD and BOD value to acceptable levels. There is now-a large body of information concerning the genetics and regulation of hydrocarbon biodegradation. The ability of microorganisms to degrade naturally occurring aromatic and aliphatic hydrocarbons is an ancient metabolic trait, but when such an organism is exposed to a xenobiotic compound, the microorganism may use degradative enzymes that have been modified by evolution or selection so that these enzymes are active towards xenobiotics. Plasmids often encode genes necessary for the degradation of hydrocarbons such as toluene (TOL+), napthalene (NAH+), octane (OCT), 2-4-dichlorophenoxyacetic acid and many others. The range of hydrocarbons that a particular microorganism can degrade can be increased through genetic manipulation by microbiologists. For example, Chakrabarty et al. have constructed a novel strain by transferring various degradative plasmids in one organism. This strain could degrade crude oil completely 22. In addition, Chakrabatty etal. have also developed a new phenotype of Pseudomonas cepacia by a process called 'plasmid assisted
~ ~ ~ ~IIxI2~ 5
7
Azide Uninoculated control control (7 days)
Changes in chemical oxygen demand (COD)~]]~,biological oxygen demand (BOD)~ , and hexane-soluble oil contents ~ during the treatment of off sludge with Rhodotorula sp.
of seven days treatment, although there was a complete removal of the hexane extractable oil fraction, the BOD and COD levels remained very high. This indicated that some of the hydrocarbons other than the saturate fraction remained undegraded. To check this possibility, oil sludge samples at different stages of treatment were fractionated into saturate, aromatic and asphaltic fractions. These fractions were then quantified gravimetrically. There was approximately 88% degradation of the saturate fraction, 63% degradation of aromatic fraction and 13% degradation of asphaltic fraction after seven clays treatment. Hence, the persistence of the asphaltic fraction along with the remaining aromatic fraction during the treatment of oil sludge could be due to differences in the susceptibility of various fractions of oil sludge to microbial degradation: the residual COD and BOD could be accounted for by these residual hydrocarbons. The sparing phenomenon could account for the failure of Rhodotorula rubra to degrade the residual aromatic and asphaltic fractions in the presence of the easily metabolizable saturate fraction. The gas chromatographic analysis of the saturate fraction of oil sludge samples at different stages of treatment reveals the preferential utilization of higher n-alkanes (n-Cl6-n-C2o) as compared to the lower n-alkanes (n-C7-n-C12) TM. This pattern points to biodegradation as the principal removal mechanism, since compounds disappeared in the order of preferential microbial utilization rather than in the order of their volatility. Such preferential utilization of n-alkanes has also been
observed during the microbial utilization of Canadian heavy oil 2°. Economic considerations
It is extremely difficult tc collect representative and comparable cost data on oil sludge disposal systems in the petroleum refining industry. Different combinations and types of oil sludges, labor and costs of construction, capital and equipment for estabrishing a complete oil sludge treatment unit make it extremely difficult to provide a general evaluation of the cost of treatment of oil sludge. Land treatment of oil sludge has been found to be not only an environmentally sound waste disposal practice, but also highly costeffective. Grove 21 compared land treatment to other methods of disposal for oil sludges. He concluded that although land farming was the cheapest alternative, the oil was not completely degraded and remained in the land. However, this drawback of the land farming technique n~ay be overcome by using highly efficient hydrocarbon degrading microorganisms to remove the oil from soil more completely. Therefore, the land treatment of petroleum industry oil sludge seems to be a logical alternative which should be seriously examined. Perspectives
Under laboratory conditions it has not proved possible to degrade oil sludge completely. Of the methods developed for the biodegradation of oil sludge, land farming for the final disposal of oil sludge has been the most successful and environmentally acceptable. However, the biodegra-
TIBTECH - AUGUST 1986
Glossary BOD and C O D - Biological oxygen d e m a n d (BOD) measures the consumption of oxygen during the biodegradation of a chemical, whereas chemical oxygen demand (COD) measures the consumption of oxygen during complete chemical oxidation of a chemical, The reduction in the values of BOD and COD can "be taken as a criteria for biodegradation. Crude oil fractions - Crude oil can be fractionated by silica gel chromatography into saturate, aromatic and asphaltic fractions by successive elution with nhexane, benzene and chloroform : methanol(1 : 1 vol./vol.), respectively. The saturate fraction consists of aliphatic hydrocarbons mainly n-alkanes, the aromatic fraction contains benzenoid hydrocarbons and the asphaltic fraction comprises hydrocarbons containing N, S, and O. m o l e c u l a r breeding '23. In this process a group of microorganisms from 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-polluted soil were incubated in a chemostat along w i t h pure bacterial cultures w h i c h harbored a variety of plasmids e n c o d i n g genes for aromatic and chloroaromatic c o m p o u n d degradation. Strong selective pressure for 2,4,5-T degradation was a p p l i e d to this microbial mixture. This apparently led to genetic transfer and recombinational events w h i c h permitted the e v o l u t i o n of 2,4,5-T degrading strain. Plasmid assisted m o l e c u l a r breeding seems to
be a reasonable explanation for the w a y in w h i c h n e w p h e n o t y p e s might evolve in natural e n v i r o n m e n t s where microbial densities and diversities are high and selective pressures are intense. S u c h a techn i q u e c o u l d also be applicable in the generation of n e w strains to degrade the c o m p l e x mixture of crude oil. Through rapid advances being m a d e in this area of genetics, a n o v e l microbial strain m a y s o m e day be constructed that will be able to degrade a w i d e array of hydrocarbons. Eventually, waste treatment systems m u s t t h e n be designed w h i c h h e l p specific microorganisms perform to expectations. A diversity of k n o w l e d g e and skills from the scientific c o m m u n i t y will be n e e d e d to design these n e w microbes and to use t h e m for clean-up.
180-209 7 Perry, J. J. (1979) Microbiol. Rev. 43, 59-72 8 Alexander, M. (1981) Science 211, 132-138 9 Llanos, C. and Kjoller, A. (1976) Oikos 27, 331-382 10 Alexander, M. (1977) Introduction to Soil Microbiology, John Wiley & Sons 11 Raymond, R. L., Hudson, J.O. and Jamison, V. M. (1980) Am. Inst. Chem. Eng. Syrup. 75, 340-356 12 CONCAWE (1980) Report No. 3/80, The Hague, Netherlands 13 Mckenna, E.J. andKallio, R. E. (1971) Proc. Natl Acad. Sci. USA 68, 1552-1554 14 Atlas, R. M. and Bartha, R. (1973) Antonie van Leewenhoek, J. Microbio]. Serol. 39, 257-271 15 Dibble, J. T. and Bartha, R. M. (1979) App]. Environ. Microbiol. 37, 729-739 16 Dibble, J. T. and Bartha, R. M. (1976) App]. Environ. Microhiol. 31,
Acknowledgement
544-550 17 Modi, V. V., Rao, N.N. and Shailubhai, K. (1980) in Management o f Environment (Patel, B., ed.), pp.
I am grateful to Dr V. V. Modi and other colleagues, w h o h a v e contributed to the w o r k described here. I thank Drs I. K. Vijay, A. K. Mattoo, A . M . Mehta, C.S. Nautiyal and Brenda Alston-Mills for their helpful suggestions about this m a n u s c r i p t and also Ms Margaret K e m p f for typing this manuscript.
References 1 Chet, I. and Mitchell, R. (1976) Nature 261,308-309 2 Parker, P. L. and Menzel, D. (1974) National Science Foundation IDOE 3 Donahue, W. H., Wang, R. T., Welch, M. and Micol, J.A. (1977)Environ. Pollut. 13, 187-202 4 Krebs, C. T. and Burns, K. A. (1977) Science 197, 484-487 5 Zobell, C. E. (1946) Bacterio]. Rev. 10, 1-49 6 Atlas, R. M. (1981) Microbiol. Rev. 45,
172-180, Eastern Wiley & Sons 18 Shailubhai, K., Rao, N. N. and Modi, V.V. (1984) App]. Microbio]. Biotechnol. 19, 437-438 19 Shailubhai, K., Rao, N. N. and Modi, V. V. (1984) Oil Petrochem. Pollut. 2, 133-136 20 Jobson, A., Cook, F. D. and Westlake, D. W. S. (1972) App]. Microbiol. 23, 1082-1089 21 Grove, G. W. (1980) in Disposal of Industrial and Oily Sludges by Land Cultivation (Shilesky, D. M., ed.), pp. 25-32, Resource Systems and Management Association 22 Friello, D. A., Mylroi, J.R. and Chakrabarty, A. M. (1976) in Proceedings of the 3rd International Biodegradation Symposium (Sharplay, J.M. and Kaplan, A.M., eds), pp. 205-214, Applied Science 23 Ghosal, D., You, I-S., Chatterjee, D. K. and Chakrabarty, A.M. (1985) Science 228, 135-142
References 1 Jensen, F. K., Blythman, H.E., Carriere, D., Casellas, P., Gros, O., Laurant, J.E., Paolucci, F., Pau, B., Poncelet, P., Richer, G., Vidal, H. and Voisin, G. A. (1982) Immuno]. Rev. 62, 185-216 2 Pirker, R., FitzerGerald, D.J., Hamilton, T.C., Ozols, R.F., Laird, W., Frankel, A. E., Willingham, M. C. and Pastan, I. (1985) Jo Clin. Invest. 76, 1261-1267
3 Vitteta, E. S. and Uhr, J.W. (1985) Annu. Rev. Immunol. 3, 197-212 4 Dorland's Illustrated Medical Diction-
ary (1974) (25th edn), p. 767, W.B. Saunders Company 5 Stedman'~s Medical Dictionary Illustrated (1979), p. 694, Williams and
Wilkins 60lsnes, S. and Phil, A. (1982) J. Pharmacol. Ther. 15, 355-381 7 Youle, R. J. and Neville, D. M. (1980) Proc. Natl Acad. Sci. USA 77,
Treatment of petroleum industry oil sludge in soil K. Shailubhai
5483-5486 8 Volkman, D. J., Ahmad, A., Fauci, A. S. and Neville, D. M. (1982) J. Exp. Med. 156, 634-639 ATEEQ AHMAD
Division o f Biochemistry, Departm e n t o f the A r m y , Walter Reed A r m y Institute of Research, Washington, DC 20307, USA.
accumulate these hydrocarbons in their fatty tissues and hence are able to transfer the toxic compounds to higher trophic levels, including humans. Therefore, the concern over chronic hydrocarbon inputs is of importance to many coastal communities. The ecologically acceptable disposal of oil sludges and other waste hydrocarbons is a major challenge confronting the petroleum industry.
Biodegradation ofoil sludge in soil Petroleum refining unavoidably generates large volumes of oil sludge. The environmentally acceptable disposal of oil sludge is a current challenge to the petroleum industry. Many soil microorganisms possess a remarkable capacity to degrade various components of crude oil. The land treatment of oil sludge - land farming - is an environmentally acceptable and economically feasible disposal method. The development of efficient hydrocarbon-degrading microorganisms and their use for cleaning oil sludge in soil are discussed. Petroleum refineries use water in many operations during the refining and cracking of heavier hydrocarbons. In the process the water gets contaminated with various chemicals. Therefore, the chemical content of petroleum refinery oil sludge consists of not only the compounds from the original crude oil stock but also those produced in fracturing processes and chemical additives used within the refining operations. The major components of oil waste include metallic and non-metallic compounds and water. The principal metallic constituents of refinery waste are zinc, chromium, vanadium, nickel, lead and copper. The main non-metallic constituents are K. Shailubhai is at the Department of Animal Sciences, University of Mary]and, College Park, MD 20742, USA.
organics such as n-alkanes, paraffins, olefins, aromatics, asphaltics, phenols and polynuclear aromatic hydrocarbons. The composition of oil sludge varies from batch to batch depending on the history of treatment and storage. Recent studies have shown that petroleum hydrocarbons can have both lethal and sublethal effects on a wide variety of marine organisms. The sublethal effects of petroleum hydrocarbons include interference with the chemotactic response of bacteria. This results in: inhibition of organic matter decomposition in sea water1; reduced photosynthetic rate in algae2; abnormal development and hatching of barnacle embryos3; and inhibition of moulting, alteration of sex ratios, and reduced resistance to environmental stress in c r a b s 4. Many fish have been shown to
(~ 1986, Elsevier Science Publishers B,V., Amsterdam 0166- 9430/86/$02.00
Soil provides a natural environment for the biodegradation of waste materials through complex physical, chemical and microbiological processes. Microorganisms present naturally in soil degrade the waste oil and produce intermediate products such as alcohols, phenols, esters, aldehydes, ketones and fatty acids. These products are ultimately converted to carbon dioxide, water and cellular materials. Although microbial assimilation is the principal means of waste degradation, the contribution of other non-biological processes (chemical and photochemical autooxidation, evaporation and volatilization) is also significant. The overall rate of biodegradation is influenced by the type of oil sludge, by the microorganisms present in soil and by the climate. The ability of microorganisms to degrade various components of petroleum has been recognized for more than a century. More than 100 species of bacteria, yeast, actinomycetes and fungi, representing 31 genera are known to attack one or more types of petroleum hydrocarbons 5. Atlas 6 listed 22 genera of bacteria and 14 genera of fungi in which hydrocarbon degradation ability had been demonstrated. Recent
TIBTECH- AUGUST1986
Table 1 Microbial genera degrading hydrocarbons in soil
Bacteria Achromobacter Aerobacillus Alcaligenes Arthrobacter Bacillus Bacterium Beijerinckia Botrytis Citrobacter Clostridium Corynebacterium Desulfovibrio Enterobacter
Escherichia Flavobacterium Gaffkya Methanobacterium Micrococcus Micromonospora Mycobacterium Pseudomonas Sarcina Serratia Spirillum Thiobacillus
studies continue to expand the list of microbial species that have been shown to be capable of degrading various components of petroleum 7'8. Table 1 lists the primary microorganisms responsible for hydrocarbon degradation in soil. The ubiquitous distribution of yeasts, fungi and bacteria in oilcontaminated environments has led to the conclusion that microorganisms play an important role in the degradation of oil in soil. Llanos and Kjoller (1976) found that the application of waste oil to soil favored the growth of organisms capable of utilizing crude oil as sole sources of carbon and energy 9. Microbial distribution patterns in a typical soil indicate that the upper soil zone has by far the largest microbial population and, thus, may be the most active zone for waste biodegradation 1°. The application of oil waste to soil causes the enrichment of specific hydrocarbon-degrading microorganisms. Raymond et aL reported an increase in the soil microbial population due to oil application from 1 x 105 to 1 x 1 0 7 microorganisms per gram of soil. 1~
Oil sludge disposal technology Oily sludges are derived from o i l water separators, tank bottoms or cleanings, air flotation treatment of waste waters, and cleanings from lagoons or oxidation ponds. A major consideration in treating oily sludges is that these materials are generated from various sources and are discharged at irregular intervals. Thus, sludge composition is highly vari-
Actinomycetes
Fungi
Yeasts
Actinomyces Endomyces Nocardia
Aspergillus Cephalosporium Cunninghamella
Candida Rhodotorula Torula Torulopsis Trichoderma Saccharomyces
able. The most common processes for handling oily sludges are gravity thickening, vacuum filtration or centrifugal dewatering, and ultimate disposal is by dumping into the oceans, into lagoons, incineration, or land farming (landfill) (Fig. 1). However, dumping into the ocean is not recommended because aquatic organisms can rapidly accumulate heavy metals and toxic organic substances. Incineration is also no longer considered to be desirable because of the energy cost and the air pollution problems that are often associated with it. The disposal of oily sludges on soil is acceptable if it can be shown that such disposal will not contaminate ground water and will not create a seepage problem. Land farming operations that use soil microorganisms to degrade and clean up the oil satisfy these requirements.
Land farming of oily sludges has been successful at refineries where sufficient area of land is available for proper decomposition of oily sludge. The technique involves spreading the oily sludge in 10-15 cm layers, allowing the sludge to dry for about one week, adding fertilizers and then discing the sludge into the soil. Most major oil refineries in the US dispose of at least a portion of their oily wastes by land farming. Land treatment is also practised in the UK, the Netherlands, Sweden, Denmark, France and New Zealand 12.
Factors affectingbiodegradation During the treatment of oil waste, the microorganisms use hydrocarbons as sources of food and energy which results in the clean-up of oil waste effluent.
Fig. 1
Oil--water separation
Dewatering
Final disposal ~,-[ Incineration
Gravity
Filtration
~[ Landfarming ~[
Oil sludge disposal technology
Lagoon Ocean disposal
T I B T E C H - A U G U S T 1986
Composition of waste The degree and rate of microbial biodegradation depends on the nature and type of hydrocarbons present in the oil waste. Among the various petroleum hydrocarbons, the n-alkanes of intermediate chain length are preferred substrates for microbial degradation, whereas highly branched ~ a l k a n e s and cyclealkanes in particular are less susceptible to microbial attack 13. Complex alicyclic compounds such as hopanes are among the most persistent components of petroleum waste 7. Aromatic hydrocarbons are also abundant in oil waste and most of them are biodegradable. However, some of them, particularly polynuclear hydrocarbons, are metabolically recalcitrant such that microbial treatment has very little effect on their fate. In addition, the viscosity, solubility and toxicity of waste constituents also exert a significant influence on their degradability. Two phenomena which need to be considered in the biodegradation process of oil sludge are 'sparing' and 'co-oxidation'. When a microorganism with a broad substrate range is offered more than one type of organic substrate, it is likely that it will not attack the substrates simultaneously, but in a definitive sequence. For example, the degradation of pristane was inhibited in the presence of the easily metabolizable substrate hexadecane ~4. The susceptibility of the various fractions of crude oil to microbial degradation is in the following order: saturate > aromatic > asphaltic fraction. Hence, during the biodegradation of oil waste, aromatic and asphaltic components are not degraded until the easily metabolizable saturated n-alkanes :are exhausted. The phenomenon of co-oxidation has also been shown to play an important role in the biodegradation of crude oil. The compounds which otherwise would not be degraded can be enzymatically attacked within petroleum mixtures because individual organisms can grow on some of the hydrocarbons within crude oil 14. Climateandnutrition Several climatic and nutritional factors also synergistically affect the
rate at which oil sludge is degraded in soil. The main factors are soil oxygen content, nutrients, moisture content, pH and temperature. The biodegradation of oil waste in soil is carried out mainly by aerobic microorganisms present in the upper layer of soil: therefore oxygen must diffuse into the soil pore space to be accessible to the microorganisms. Anaerobic microoganisms will also degrade oil sludge in an anaerobic atmosphere, with nitrates, nitrites and sulfates replacing oxygen as oxidants. The most rapid biodegradation rates occur in the presence of oxygen; to achieve maximum waste soil interaction and to provide sufficient air (oxygen), the soil is tilled frequently. Biodegradation of hydrocarbons requires the presence of nitrogen, phosphorus and potassium, as well as lower levels of zinc, calcium, manganese, magnesium, iron and sulfur; the latter elements are essential for microbial metabolism. The nutrients can be provided by application of fertilizers to the soil and this is necessary to maintain or enhance the biodegradation rate. The amount of oil waste degraded was found to double with the addition of fertilizers 1~. Of the minerals, iron has been considered as potentially limiting for petroleum biodegradation even in sea water containing nitrogen and phosphorus 16. Extremes of very wet or very dry soil markedly reduce waste biodegradation rates. Saturated soil may affect oil degradation due to low supply of oxygen whereas very dry conditions may affect the degradation rates due to insufficient moisture contents 16. Temperature and pH are other factors which affect the biodegradation rates significantly. For example, very acidic or very alkaline soil pH will retard the microbial growth and activity. Temperature also affects the abiotic removal of oil waste from soil.
Enhanced oil sludge degradation The preceding discussion suggests that the success of oil sludge biodegradation by land farming is influenced by a complex mix of chemical, physical and biological factors. Though oil sludge biodegradation by land farming has been
successful, important parameters, such as the effect of tillage, soil texture and fluctuation in temperature during a 24 hour cycle have not been studied, Moroever, the oil sludge biodegradation in soil is a very slow process. The limitations, side effects, and high expense of traditional clean-up technology has stimulated interest in unconventional alternatives, such as the use of hydrocarbon-degrading microorganisms for clean-up or increasing the biodegradation rate artificially by providing optimum conditions. It appears possible to enhance oil sludge biodegradation, in an enclosed, temperature controlled aerobic system by inoculation of highly efficient oil degrading microorganisms, along with some mineral nutrients. Unlike 'land farming', this method of oil sludge disposal is less dependent on favorable weather conditions and is equally amenable to manipulation. This approach has been used successfully to develop a treatment method for the biodegradation of oil sludge: Rhodotorula rubra, a yeast strain found to be capable of degrading a wide range of aliphatic and aromatic hydrocarbons, was inoculated in oil sludge along with some mineral salts in a temperature controlled (28°C) stainless steel fermentor 17'18. Aeration was provided by compressed filter-sterilized air. Under similar conditions uninoculated controls and controls to which azide was added to prevent microbial activity were also incubated. Samples were removed for analysis after seven days. As a result of biodegradation, there was 50-60% decrease in the chemical oxygen demand (COD) and 80-90% decrease in the biological oxygen demand (BOD) (Fig. 2). (The decrease in COD and BOD values can be taken as criteria for the degradation of hydrocarbons present in oil sludge.) There was also a 40% decrease in BOD and 14 OYodecrease in COD in the uninoculated control. This may reflect the activities of the microflora within the oil sludge itself. There was also some decrease in BOD (4.7%) and COD (8.0%) values in the azideadded control, which was attributed to the weathering of the volatile components of crude oil. At the end
T I B T E C H - A U G U S T 1986
--
Fig. 2
×10 0
~ 1
3 Time (days)
~ ~3~_
dation rate in land farming is very slow and is dependent on biological and climatic factors. These limitations can be overcome by mixing highly efficient hydrocarbon-degrading microorganisms in soil and by providing optimum conditions for biodegradation of crude oil. For instance, it has been shown that, by using Rhodotorula rubra, a very efficient hydrocarbon degrading yeast, the oil sludge under laboratory conditions could be degraded to an acceptable level within seven days of treatment. However, these findings will need validation and possible adjustments on the industrial scale. Nevertheless, it may greatly reduce and simplify oil sludge treatment on a larger scale. Future developments on the oil sludge treatment process will require a close collaboration between researchers in microbiology and bioengineering technology to implement full scale processes. Another aspect which requires further work is the development of microorganisms which can degrade various fractions of oil sludge completely in order to bring down its COD and BOD value to acceptable levels. There is now-a large body of information concerning the genetics and regulation of hydrocarbon biodegradation. The ability of microorganisms to degrade naturally occurring aromatic and aliphatic hydrocarbons is an ancient metabolic trait, but when such an organism is exposed to a xenobiotic compound, the microorganism may use degradative enzymes that have been modified by evolution or selection so that these enzymes are active towards xenobiotics. Plasmids often encode genes necessary for the degradation of hydrocarbons such as toluene (TOL+), napthalene (NAH+), octane (OCT), 2-4-dichlorophenoxyacetic acid and many others. The range of hydrocarbons that a particular microorganism can degrade can be increased through genetic manipulation by microbiologists. For example, Chakrabarty et al. have constructed a novel strain by transferring various degradative plasmids in one organism. This strain could degrade crude oil completely 22. In addition, Chakrabatty etal. have also developed a new phenotype of Pseudomonas cepacia by a process called 'plasmid assisted
~ ~ ~ ~IIxI2~ 5
7
Azide Uninoculated control control (7 days)
Changes in chemical oxygen demand (COD)~]]~,biological oxygen demand (BOD)~ , and hexane-soluble oil contents ~ during the treatment of off sludge with Rhodotorula sp.
of seven days treatment, although there was a complete removal of the hexane extractable oil fraction, the BOD and COD levels remained very high. This indicated that some of the hydrocarbons other than the saturate fraction remained undegraded. To check this possibility, oil sludge samples at different stages of treatment were fractionated into saturate, aromatic and asphaltic fractions. These fractions were then quantified gravimetrically. There was approximately 88% degradation of the saturate fraction, 63% degradation of aromatic fraction and 13% degradation of asphaltic fraction after seven clays treatment. Hence, the persistence of the asphaltic fraction along with the remaining aromatic fraction during the treatment of oil sludge could be due to differences in the susceptibility of various fractions of oil sludge to microbial degradation: the residual COD and BOD could be accounted for by these residual hydrocarbons. The sparing phenomenon could account for the failure of Rhodotorula rubra to degrade the residual aromatic and asphaltic fractions in the presence of the easily metabolizable saturate fraction. The gas chromatographic analysis of the saturate fraction of oil sludge samples at different stages of treatment reveals the preferential utilization of higher n-alkanes (n-Cl6-n-C2o) as compared to the lower n-alkanes (n-C7-n-C12) TM. This pattern points to biodegradation as the principal removal mechanism, since compounds disappeared in the order of preferential microbial utilization rather than in the order of their volatility. Such preferential utilization of n-alkanes has also been
observed during the microbial utilization of Canadian heavy oil 2°. Economic considerations
It is extremely difficult tc collect representative and comparable cost data on oil sludge disposal systems in the petroleum refining industry. Different combinations and types of oil sludges, labor and costs of construction, capital and equipment for estabrishing a complete oil sludge treatment unit make it extremely difficult to provide a general evaluation of the cost of treatment of oil sludge. Land treatment of oil sludge has been found to be not only an environmentally sound waste disposal practice, but also highly costeffective. Grove 21 compared land treatment to other methods of disposal for oil sludges. He concluded that although land farming was the cheapest alternative, the oil was not completely degraded and remained in the land. However, this drawback of the land farming technique n~ay be overcome by using highly efficient hydrocarbon degrading microorganisms to remove the oil from soil more completely. Therefore, the land treatment of petroleum industry oil sludge seems to be a logical alternative which should be seriously examined. Perspectives
Under laboratory conditions it has not proved possible to degrade oil sludge completely. Of the methods developed for the biodegradation of oil sludge, land farming for the final disposal of oil sludge has been the most successful and environmentally acceptable. However, the biodegra-
TIBTECH - AUGUST 1986
Glossary BOD and C O D - Biological oxygen d e m a n d (BOD) measures the consumption of oxygen during the biodegradation of a chemical, whereas chemical oxygen demand (COD) measures the consumption of oxygen during complete chemical oxidation of a chemical, The reduction in the values of BOD and COD can "be taken as a criteria for biodegradation. Crude oil fractions - Crude oil can be fractionated by silica gel chromatography into saturate, aromatic and asphaltic fractions by successive elution with nhexane, benzene and chloroform : methanol(1 : 1 vol./vol.), respectively. The saturate fraction consists of aliphatic hydrocarbons mainly n-alkanes, the aromatic fraction contains benzenoid hydrocarbons and the asphaltic fraction comprises hydrocarbons containing N, S, and O. m o l e c u l a r breeding '23. In this process a group of microorganisms from 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-polluted soil were incubated in a chemostat along w i t h pure bacterial cultures w h i c h harbored a variety of plasmids e n c o d i n g genes for aromatic and chloroaromatic c o m p o u n d degradation. Strong selective pressure for 2,4,5-T degradation was a p p l i e d to this microbial mixture. This apparently led to genetic transfer and recombinational events w h i c h permitted the e v o l u t i o n of 2,4,5-T degrading strain. Plasmid assisted m o l e c u l a r breeding seems to
be a reasonable explanation for the w a y in w h i c h n e w p h e n o t y p e s might evolve in natural e n v i r o n m e n t s where microbial densities and diversities are high and selective pressures are intense. S u c h a techn i q u e c o u l d also be applicable in the generation of n e w strains to degrade the c o m p l e x mixture of crude oil. Through rapid advances being m a d e in this area of genetics, a n o v e l microbial strain m a y s o m e day be constructed that will be able to degrade a w i d e array of hydrocarbons. Eventually, waste treatment systems m u s t t h e n be designed w h i c h h e l p specific microorganisms perform to expectations. A diversity of k n o w l e d g e and skills from the scientific c o m m u n i t y will be n e e d e d to design these n e w microbes and to use t h e m for clean-up.
180-209 7 Perry, J. J. (1979) Microbiol. Rev. 43, 59-72 8 Alexander, M. (1981) Science 211, 132-138 9 Llanos, C. and Kjoller, A. (1976) Oikos 27, 331-382 10 Alexander, M. (1977) Introduction to Soil Microbiology, John Wiley & Sons 11 Raymond, R. L., Hudson, J.O. and Jamison, V. M. (1980) Am. Inst. Chem. Eng. Syrup. 75, 340-356 12 CONCAWE (1980) Report No. 3/80, The Hague, Netherlands 13 Mckenna, E.J. andKallio, R. E. (1971) Proc. Natl Acad. Sci. USA 68, 1552-1554 14 Atlas, R. M. and Bartha, R. (1973) Antonie van Leewenhoek, J. Microbio]. Serol. 39, 257-271 15 Dibble, J. T. and Bartha, R. M. (1979) App]. Environ. Microbiol. 37, 729-739 16 Dibble, J. T. and Bartha, R. M. (1976) App]. Environ. Microhiol. 31,
Acknowledgement
544-550 17 Modi, V. V., Rao, N.N. and Shailubhai, K. (1980) in Management o f Environment (Patel, B., ed.), pp.
I am grateful to Dr V. V. Modi and other colleagues, w h o h a v e contributed to the w o r k described here. I thank Drs I. K. Vijay, A. K. Mattoo, A . M . Mehta, C.S. Nautiyal and Brenda Alston-Mills for their helpful suggestions about this m a n u s c r i p t and also Ms Margaret K e m p f for typing this manuscript.
References 1 Chet, I. and Mitchell, R. (1976) Nature 261,308-309 2 Parker, P. L. and Menzel, D. (1974) National Science Foundation IDOE 3 Donahue, W. H., Wang, R. T., Welch, M. and Micol, J.A. (1977)Environ. Pollut. 13, 187-202 4 Krebs, C. T. and Burns, K. A. (1977) Science 197, 484-487 5 Zobell, C. E. (1946) Bacterio]. Rev. 10, 1-49 6 Atlas, R. M. (1981) Microbiol. Rev. 45,
172-180, Eastern Wiley & Sons 18 Shailubhai, K., Rao, N. N. and Modi, V.V. (1984) App]. Microbio]. Biotechnol. 19, 437-438 19 Shailubhai, K., Rao, N. N. and Modi, V. V. (1984) Oil Petrochem. Pollut. 2, 133-136 20 Jobson, A., Cook, F. D. and Westlake, D. W. S. (1972) App]. Microbiol. 23, 1082-1089 21 Grove, G. W. (1980) in Disposal of Industrial and Oily Sludges by Land Cultivation (Shilesky, D. M., ed.), pp. 25-32, Resource Systems and Management Association 22 Friello, D. A., Mylroi, J.R. and Chakrabarty, A. M. (1976) in Proceedings of the 3rd International Biodegradation Symposium (Sharplay, J.M. and Kaplan, A.M., eds), pp. 205-214, Applied Science 23 Ghosal, D., You, I-S., Chatterjee, D. K. and Chakrabarty, A.M. (1985) Science 228, 135-142