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Revista de Microbiologia

Print version ISSN 0001-3714

Rev. Microbiol. vol. 29 n. 4 São Paulo Oct./Dec. 1998 



Adriana F. P. Ururahy1; Marcus D. M. Marins2; Ronalt L. Vital2; Irene Therezinha Gabardo2; Nei Pereira Jr.1* 
1Departamento de Engenharia Bioquímica, Escola de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil; 2CENPES - Centro de Pesquisas Desenvolvimento da PETROBRAS, Rio de Janeiro, RJ, Brasil.

Submitted: October 15, 1997; Returned to authors for corrections: April 16, 1998;
Approved: January 20, 1999




Large amounts of oily sludge are generated as residues by the oil industry, representing a real problem for refineries. This work studied the technical viability of treating oily sludge biologically, through stimulation of native microorganisms, at bench scale. Such microorganisms were able to grow in a medium containing oily sludge as the only carbon and energy sources. Two oily sludge concentrations were studied, 5% (v/v) and 10% (v/v), with a C:N ratio of 100:1. Higher microbial populations were observed in the first case. Substrate inhibition and/or toxic effect took place in the second case. The importance of aeration on the microbial activity and on the biodegradation of the residue was ascertained. In terms of n-paraffins, pristane and phytane consumption, maximum global efficiency of 76.9% (w/w) was achieved, in a medium containing 5% (v/v) of oily sludge. Bacteria of the genus Pseudomonas predominated. Two yeast species were also identified and two filamentous fungi were isolated.

Key words: petroleum waste, biodegradation, aeration




The oil industry is responsible for the generation of high amounts of oily and viscous residues, which are formed during production, transportation and refining. Such residues, called oily sludges, are basically composed of oil, water, solids, and their characteristics, such as varied composition, make their reutilization very difficult, and confer on them high recalcitrance.

The marked stability of the multiphase system is due to the adsortion of oil on the solid particles, producing a highly protective layer, since they tend to deposit in the bottom of tanks. Additionally, the polar fractions promote charge repulsion, imparing the formation of a homogeneous phase (15). From a chemical point of view, this recalcitrance can be ascribed to the presence of aromatic, polycyclic aromatic hydrocarbons (PAHs) and complex compounds with a very high molecular weight, such as asphaltenes. In addition to that, some of these compounds act as solvent of microbial membranes.

It is estimated that, approximately, 1% of the total oil processed in a refinery in Rio de Janeiro (Brazil) is discarded as oily sludge. This has been accumulated in storage tanks for several years.

Due to the high energy costs, the potential risk of air pollution and the persistence of PAHs, incineration is not recommended. Similarly, inadequate disposal of a such very toxic residue in landfills, encourage the search of other alternatives. Biotreatment can be applied, using the following methods: Composting, Landfarming and Biopile (9). All of them exploit soil biodiversity; however they have the disadvantage of needing long process times and there is the risk of contaminating air and aquifers by leaching. They also demand large areas and are affected by climate. An interesting alternative to circumvent these problems is the use of a bioreactor, since optimum process conditions can be easily controlled, allowing higher quality final effluent in shorter times. However they might have high costs. Taking advantage of such potential, the Valero Refining Company developed, for the first time on an industrial scale, a process using a bioreactor for treating oily sludge. One of the refineries, Corpus Christi, published an economic study, comparing biotreatment with incineration for a capacity of 2000 t/year residue. In a non-optimized system, in terms of oxygen utilization, chemical cost and waste quality, a lower cost for the biotreatment was achieved, making it economically competitive and environmental friendly (16).

Bacteria, yeasts and filamentous fungi have been reported as transforming agents because of their ability to degrade a wide variety of xenobiotic substances, commonly found in wastes from the oil industry. Being able to use these substances as the only carbon and energy source, microorganisms are powerful alternatives to conventional methods in resolving environmental problems. Prince and Sambasivam (17) point out several advantages of biological treatment: mineralization promotes permanent destruction of these residues, eliminating the hazard of further contamination and leading to high acceptance by public opinion. It can also be coupled with other processes, increasing global treatment efficiency. Optimization can be achieved by exploiting several biological phenomena, such as: microbial organization into consortia (12,14), cometabolic actions (7,13), bioaugmentation (18, 21), capacity of adaptation (20), and the possibility of genetic manipulation (8). Moreover, adhesion mechanisms and water/oil interfacial area are of great importance, affecting directly the uptake of hydrocarbon by cells and the extent of biodegradation (5,19).

This work encompasses the study of the effect of aeration on oily sludge biodegradation by native microorganisms, previously adaptated in a medium containing oily sludge as the only carbon and energy source.



Oily sludge characterization: The characterization was performed in raw oily sludge, aqueous and organic phases. Total nitrogen and ion concentration were determined in the aqueous phase by Chemiluminescence (4) and by HPIC (Dionex, HPIC-AS4A column), respectively. Total nitrogen concentration was also evaluated in the organic phase by a modified Kjeldahl Method (1). Paraffins, aromatic hydrocarbons, resins and asphaltenes analyses were performed by TLC-FID (Iatroscan MK-5), and total polycyclic aromatic hydrocarbons by UV absortion in the wave length range of 220 - 450 nm (10). Distillation range (3) and dynamic viscosity (2) were also determined.

Biomass: The inoculum size and microbial growth were evaluated by total enumeration of colony forming units (CFU), in TSA medium, whose composition is as follows (in g/l): glucose, 10; yeast extract, 2; meat peptone, 5; agar, 15; NaCl, 5 (pH=7.2).

Isolation: The Streak plate method was used with different media (TSA and MacConkey for bacteria, and Sabouraud for yeasts and filamentous fungi).

Identification: Bacteria were the only group which, so far, have been identified, using the Identification Kit API 20 NE, manufactured by BioMérieux.

Inoculum preparation: A medium containing 5%(v/v) of oily sludge, 30 ml of mineral medium (Bushnell-Haas - g/l: MgSO4, 0.2; CaCl2, 0.02; KH2PO4, 1.0; K2HPO4, 1.0; NH4NO3, 1.0; FeCl3, 0.05; pH=7.0 ) and 17.5 ml of water, in a 500 ml conical flask, was placed in a rotary shaker (170 rpm, 30oC) for two days. 10 ml of this medium were transfered to a flask, containing the medium, and again incubated. This procedure was repeated, every two days for six days. The final culture was used to start the biodegradation process.

Experimental procedure: The biodegradation essays were carried out with two different oily sludge concentrations (v/v): 5% (MA) and 10% (MB). Both media were prepared to give a C:N ratio of 100:1. Water and mineral medium were added in the same proportion as in the medium for inoculum preparation. Evaporation was evaluated through a control, containing 3g/L of sodium azide (MA* and MB*). The experiments were performed in flasks, closed with caps, through which they could be saturated with oxygen every 24 hrs for 5 days. The system was shaken for two days at 150 rpm. The microbial activity was estimated by CO2 emission, over 42 days, employing gas chromatography (HP 5890II, Porapack - 2.5 m x 1/8" column). n-paraffins (nC-10 to nC-34), pristane and phytane concentrations (in µg/ml) were determined by GC, employing DB5 - 30m x 0.32mm column (J&W). The biodegradation efficiency based on respiration rate was calculated as accumulated CO2 during the whole time period, using by the following expression:

EB(%) = CO2 bio x 100/Ci

CO2 bio = 2 x CO2 total - CO2 control


EB = Efficiency of biodegradation (% µmols/µmols)
CO2 bio = µmols of CO2 produced by microbial activity
CO2 total = µmols of total CO2 accumulated
Ci = µmols of CO2 equivalent to initial carbon (theoretical value)
CO2 control = µmols of CO2 accumulated in the control (abiotic system)

The biodegradation efficiency was also evaluated by the decrease in the concentration of n-paraffins, pristane and phytane.



Before the adoption of any technological strategy for treating a residue, its characterization is required. Table 1 displays the composition and main physical properties of the oily sludge from Duque de Caxias Refinery (REDUC).


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Table 1: Oil sludge characterization


It can be seen that the oily sludge possesses limited amounts of nitrogen and phosphate, indicating the necessity for fortifying the media with nutrients (11). Most of the nitrogen is not available, since it is part of complex structures, relatively inaccessible to microorganisms.

Although the concentration of asphaltenes is not very high, the predominance of aromatics and the high concentration of polycyclic aromatic hydrocarbons, usually found in this kind of residue (6), confer on it high toxicity.

The range of distillation (289 to 550oC) shows the preponderance of heavy compounds of considerable stability, low solubility in water and consequently marked recalcitrance. This high viscosity, which does not hamper microbial attack on the oil, points to the importance of mechanical agitation.

The microbial capacity to grow, using the oily sludge as sole source of carbon and energy can be observed in Fig. 1, which shows the daily CO2 production versus time. It is clear that the interruption of oxygen feed to the media (points indicated by arrows), led to a dramatic decrease in the rate of CO2 evolution, denoting a significant decrease in microbial activity. Between the 23rd and 25th days, for instance, the rate of gas evolution decreased from 2.5 µmol/day to 0.8 µmol/day.


0004i03.jpg (24844 bytes)

Figure 1 - Evolution rates of CO2 in the media MA - 5% (v/v) of oily sludge (·), MB - 10% (v/v) of oily sludge (u) and controls - MA* (-) and MB* (+). 
Arrows = air feed interruption


Low biodegradation efficiencies were achieved for 10% sludge (v/v) (EB21days=8.9% and EB42days =20.7%). This may be due to substrate inhibition and/or toxic effects. The biodegradation efficiencies for 5% (v/v) were higher (EB21days=17.4% and EB42days =39.0%). These results were corroborated by higher microbial population in the last case (Fig. 2).


0004i04.jpg (19889 bytes)

Figure 2 - Microbial growth in the media MA - 5% (v/v) of oily sludge (n) and MB - 10% (v/v) of oily sludge(o). 
Microbial population (N) in CFU/ml.


The uptake of paraffins, particularly pristane and phytane, resulted in higher biodegradation of both compounds in medium MA (33.7% w/w and 10.2% w/w, respectively) and low utilization in medium MB (0.0% w/w and 0.2% w/w). According to Watkinson and Morgan (22), pristane is widely employed as an internal standard for analyses of hydrocarbon samples, as it has a considerable degree of persistence. Thus, the significant consumption of this substance shows the potential of the biotreatment system. The consumption of paraffins as a whole, for medium MA, can be seen in the chromatograms in Figs 3 (initial), 4 (intermediate) and 5 (final). There is a decrease in the peaks of a wide range of compounds, from nC-10 to nC-34. The global efficiences of biodegradation were 76.9% (w/w) and 50.6% (w/w) in medium MA and MB, respectively.


0004i05.gif (3248 bytes)

Figure 3 - Medium MA: initial analysis of n-paraffins, pristane and phytane (t=0)



0004i06.gif (2274 bytes)

Figure 4 - Medium MA: intermediate analysis of n-paraffins, pristane and phytane (t=21 days)



0004i07.gif (2210 bytes)

Figure 5 - Medium MA: final analysis of n-paraffins, pristane and phytane (t=42 days)


Because of analytical limitations, PAHs were not determined, but attempts will be made, in future experiments, to overcome this problem.

The identification of native bacteria showed a predominance of the genus Pseudomonas, which was expected since this genus has been commonly found in affected areas (9). So far, the folowing bacteria species have been identified: Pseudomonas cepacia, Pseudomonas aureofaciens, Pseudomonas picketti, Flavobacterium indologenes, Xanthomonas maltophilia and Ochrobactrum anthropi. Two yeast species, Candida tropicalis and Rhodotorula mucilaginosa, were also identified and two filamentous fungi were isolated, although they have not been identified yet. Once more, the importance of aeration became evident, since all bacterial species are strictly aerobic microorganisms. Such identification will allow the adoption of strategies in the optimization of oily sludge biotreatment.



This work was supported by PETROBRAS, through grants to the first author and for laboratory infrastructure facilities.




Efeito da aeração sobre a biodegradação de resíduo de petróleo

Grandes quantidades de borra oleosa são geradas como resíduos pela indústria do petróleo, representando um problema real para as refinarias. Neste trabalho foi estudada a viabilidade técnica do tratamento biológico de borra oleosa, conduzido a partir do estímulo de microrganismos nativos, em escala de bancada. Tais microrganismos foram capazes de crescer em meio contendo borra oleosa como única fonte de carbono e de energia. Duas concentrações deste resíduo foram estudadas, 5% (v/v) e 10% (v/v), para uma relação C:N de 100:1. Maiores densidades microbianas foram observadas na primeira condição. Por outro lado, inibição pelo substrato e/ou efeito tóxico ocorreram na segunda condição. Foi comprovada a importância da aeração sobre a atividade microbiana, assim como sobre a biodegradação do resíduo. Em termos de consumo de n-parafinas, pristano e fitano, a eficiência global máxima atingida foi de 76,9% (p/p), em meio contendo 5% (v/v) de borra oleosa. O procedimento de identificação mostrou a predominância de bactérias do gênero Pseudomonas e de leveduras dos gêneros Candida e Rhodotorula. Dois fungos filamentosos também foram isolados, estando, no momento, sujeitos a procedimentos de identificação.

Palavras-chave: borra oleosa, biodegradação, aeração




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* Corresponding author. Mailing address: Departamento de Engenharia Bioquímica, Escola de Química, Universidade Federal do Rio de Janeiro, CEP 21941-590, Rio de Janeiro, RJ, Brasil. Fax: (+5521) 590-4991. E-mail:

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