Use of selected autochthonous soil bacteria to enhanced degradation of hydrocarbons in soil

Use of selected autochthonous soil bacteria to enhanced degradation of hydrocarbons in soil

Environmental Pollution 67 (1990) 249-258 Use of Selected Autochthonous Soil Bacteria to Enhance Degradation of Hydrocarbons in Soil G. I. Vecchioli...

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Environmental Pollution 67 (1990) 249-258

Use of Selected Autochthonous Soil Bacteria to Enhance Degradation of Hydrocarbons in Soil

G. I. Vecchioli, M. T. Del P a n n o & M. T. Painceira Area Microbiologia, Facuhad de Ciencias Exactas, Universidad Nacional de La Plata, 47 y 115 (1900) La Plata, Argentina (Received 12 March 1990; accepted 17 May 1990)

ABSTRACT A mixed population of soil hydrocarbon degrading bacteria was used to accelerate the biodegradation of a petrochemical waste. An aromatic hydrocarbon storage tank bottom was mixed with soil (10% w/w). After a month 43% of the hydrocarbons were degraded in uninoculated and in fertilized soil, while 65% were degraded in inoculated soil. Nutrient supplemented vermiculite seems to be a good possibility to produce effective hydrocarbon degrading inoculants.

INTRODUCTION The rate of microbial decomposition of organic compounds in soils is a function of three variables: (a) the availability of the chemicals to the microorganisms that can degrade them; (b) the quantity of these microorganisms; and (c) the activity level of these organisms. Factors such as contents of organic matter and clay, moisture level, temperature, pH, aeration and nutrient status, are of importance as moderators and driving factors (Torstensson, 1988). One way to enhance biodegradation of organic compounds is to inoculate the environment with microorganisms known to metabolize the chemicals readily. Both successes and failures have been reported when species capable of destroying organic compounds in culture are added to samples of natural environment (Goldstein et al., 1985). In the environment the introduced 249 Environ. Pollut. 0269-7491/90/$03'50 © 1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain


G. L Vecchiofi, M. 7+. Del Panno, M. T. Pa&ceira

species may face competition, predation or parasitism. Any of these interactions could account for the inability of introduced microorganisms to survive (Walter et al., 1987). The potential of hydrocarbon mixtures to be eliminated at a higher rate from soil when hydrocarbon degrading bacteria are added to the soil is a much disputed matter. A general criticism of the seeding approach is that the use of an allochthonous microbial population may not be necessary or effective in most cases. Also, most isolates implicated in petroleum hydrocarbon biodegradation are gram-negative, non-sporeforming bacteria. These are difficult to store in large quantities in a manner that preserves their viability (Bartha, 1986). Despite these criticisms there exists an active marketing by various companies, of microbial inoculation materials that are supposed to increase hydrocarbon degradation in soils. (Johnson et al., 1985; Bartha, 1986). This study was undertaken to evaluate the possibilities of laboratorygrown hydrocarbon degrading bacteria to increase biodegradation of hydrocarbons in soil. Bacterial population was determined in inoculated and uninoculated soil, that received a complex mixture of aromatic hydrocarbons from a petrochemical storage tank bottom. The influence of autochthonous soil inoculants and of fertilizers on biodegradation of the hydrocarbons was considered. The method described by Graham-Weiss et al. (1987) using nutrient supplemented vermiculite was examined as a possibility for inoculant production.


Fresh clay loam soil was collected from a plot adjacent to a facility where petrochemical wastes are landtreated. It was sieved through 2 mm diameter openings without complete air-drying. A 100 g oven-dried (105°C) sample of this soil was added to a 600 ml flask. As required it was mixed thoroughly with 10% (w/w) of a tank bottom hydrocarbon mixture. All the hydrocarbons in the mixture were aromatics of more than 11 carbons. Duplicates of the following treatment systems were prepared: (a) Soil+hydrocarbon mixture (b) Soil + hydrocarbon mixture + bacterial inoculum (c) Soil + hydrocarbon mixture + fertilizers

Enhancing degradation o.1"hydrocarbons in soil


The fertilizers used were NH4NO 3 (60/amol/g) and K2HPO 4 (5#mol/g) (Bartha & Song, 1986). Soil without hydrocarbons was used as the untreated control. Control systems for determining abiotic hydrocarbon losses were prepared with 2% HgCI2 on a dry soil basis. All samples were moistened to 50% of the soil water holding capacity and the flasks were covered with plastic screw cups to prevent evaporation. Incubation was in the dark at 30°C. Flasks were opened for aeration at five day intervals and evaporated water, monitored by weight loss, was replenished as it was needed to maintain conditions favorable for biodegradation. On day 0 and at each subsequent sampling point (8, 16, 34 and 57) duplicate samples (1 g) from treatments a and b were diluted serially (10-fold steps) in distilled water and transferred aseptically onto appropriate agar medium for total viable and hydrocarbon degrading bacteria enumeration. Soil hydrocarbon content was determined on day 0 and after 15 and 34 days in the three treatment systems.

Media and growth conditions For growth in liquid medium on hydrocarbons, a mineral salts medium was used as a carbon source (LMM) (5g of NaC1, 1 g of K2HPO4, 1 g of NH4H2PO 4, 1 g of (NH4)SO4, 0"2 g of MgSO 4 . 7H20 and 3 g of K N O 3 per liter of bidistilled water, pH 7) supplemented with 1% filter sterilized hydrocarbon source (Solari & Painceira, 1976). The cultures were incubated at 30°C on a rotary shaker. For enumeration of hydrocarbon degrading microorganisms, plates containing L M M solidified with 1.5% Bacto agar (Difco) were seeded. Sterile filter disks were then saturated with the hydrocarbon mixture and placed on the inner surface of the petri dish cover (Painceira & Molo, 1988; Vecchioli et al., 1988). Plates were incubated at 30°C for 7 days. Plate Count agar (Difco) (PC) was used for determination of total viable counts (Brown et al., 1983). Plates were incubated at 30°C for 48h.

Source of inocula The microbial population was obtained from soil collected at an active petrochemical waste landtreatment unit. An enrichment culture was obtained by adding 1 g of soil to 100ml of hydrocarbon supplemented LMM. This enrichment culture was transferred every three days to fresh medium. Several transfers were made to be sure it was growing on the hydrocarbon source. After one transfer on PC agar slants a suspension was made and used as inoculum.


G. L Vecchioli, M. T. Del Panno, M. T. Painceira

Analytical methods Each soil sample was extracted twice by exhaustively shaking with ethylether. Undecane was added as internal standard. Analysis of residual total hydrocarbons was performed by gas chromatography. A HewlettPackard 5880A was equipped with a splitless injector and a F I D detector, and linked with a Level Four integrator. The fused silica capillary column was a 1 2 m x 0 . 2 m m , cross-linked column (Hewlett-Packard 19091A-101). Temperature was programmed from 30 to 200°C, 8°C/min. The carrier gas was H 2.

Production of inoculants Three different bacterial strains isolated from the enrichment culture were used as inoculum. Identification of each strain was based on the scheme outlined in Bergey's Manual of Systematic Bacteriology (Krieg & Holt, 1984). To confirm the ability of the strains to grow on the hydrocarbon mixture, they were grown in L M M and SMM with the hydrocarbons as sole carbon source. The strains were stored at 4°C in hydrocarbon supplemented LMM. For preparing vermiculite-based inoculants each isolated bacterium was cultured in nutrient broth (Difco). Vermiculite particles ( < 4 m m ) were autoclaved with 1.5 ml of nutrient broth per gram of dry vermiculite in screw cup flasks. A total of 10 6 to 107 bacteria was added per gram of vermiculite. The inoculant was stored at room temperature (22-25°C). A one gram portion of the inoculant was removed from the container at the start of the experiment and after 21 and 60 days of storage, and suspended in 10ml distilled water. The suspended samples were serially diluted and plated on PC for determination of bacterial population and on SMM to enumerate the bacteria that kept their ability of growing on the hydrocarbons as carbon source. RESULTS A N D DISCUSSION

Effect of sludge addition on soil bacteria population In the untreated control, viable counts (Fig. IA) were in the usual values for fertile soils (Alexander, 1977). Hydrocarbon degrading bacteria were found in the 105 range (Fig. 1B). This value confirms the presence of hydrocarbon degraders in natural soils (Odu, 1972). The addition of a 10% loading rate of an aromatic hydrocarbon mixture increased the number of total viable soil bacteria and hydrocarbon


Enhancing degradation o f hydrocarbons in soil 10


~3 "+ 2













9 8.5 -~ ~3 g







6.56 ""--e-.---














Fig. 1. Enumeration of soil bacteria in (1) untreated soil, (2) uninoculated soil (soil+hydrocarbons), and (3) inoculated soil (soil+hydrocarbons+bacteria). A: Total viable bacteria counts. B: Hydrocarbon degraders counts.


G. L Vecchioli, M. T. Del Panno, M. T. Painceira

degraders above the counts in untreated soil (Fig. l, A and B). The same effect has been observed previously (Odu, 1972; Brown~ et al., 1983) for different hydrocarbon mixtures. The effect of the hydrocarbons addition was more evident on the hydrocarbon degrading bacteria than on the total soil viable bacteria, but it was not until the second week of treatment that maximum counts were reached in both cases. In a previous experiment (data not shown) were a most favorable 1% hydrocarbons were added, the maximum number of degraders was reached in a week. These results agree with the general consideration that a 5% hydrocarbon content in soil usually brings about the optimum degradation for several hydrocarbon complex mixtures (Dibble & Bartha, 1979; Brown et al., 1983). The previous assumptions suggested to us that a 10% loading rate may limit hydrocarbon biodegradation initially and will also provide enough concentration of hydrocarbons to permit the inoculum to survive, hence a 10% loading rate was used for the inoculation experiment. Effect of inoculation on soil population

A mixed inoculum was used for the inoculation experiment since it would probably be more efficient in the breakdown of the hydrocarbon mixture as suggested by Brown (1983); Cooper & Hedrick (1976). The ability of the inoculum to survive was checked by comparing bacterial counts in both inoculated and uninoculated samples. As shown in Fig. l, A and B, the inoculation provides a high number of hydrocarbon degrading bacteria from the start of the experiment. For the first 16 days degraders remain in a higher number in inoculated soil than in uninoculated soil. Later on, differences in degraders counts between both soils were narrower. Nevertheless, counts in inoculated soil are always above those in the uninoculated soil. The presence of a naturally incremented soil microflora because of the hydrocarbons addition did not appear to affect the ability of the inoculated strains to survive. Apparently, the inoculated hydrocarbon degrading bacteria are able to compete with the soil microflora and survive at least during the early stages of the decomposition process. Effect of inoculation and fertilization on degradation

Analytical results confirm what could be suspected from the higher degraders counts achieved in the inoculated soil. As is shown in Table 1 inoculation increased hydrocarbon biodegradation 22% over that observed in the uninoculated soil. No difference was noted between the uninoculated

Enhancing degradation of hydrocarbons in soil


TABLE 1 Degradation of Hydrocarbon Mixture with Time

Time (days)

Product degraded (in per cent (![amount added) Uninoculated

0 15 34

0 29-41 43-52

Inoculated 0 48"80 65.47

Fertilized 0 25"90 44"20

Poisoned 0 -5

and the fertilized samples in the same period. The hydrocarbon loss in poisoned soil (HgC12 added) could be the result of volatilization. At the end of the experiment both total viable and degraders counts in inoculated and uninoculated soils remain above those in untreated soil. This appreciation is in accordance with the presence of undegraded hydrocarbons in both soils. Our study shows the effectiveness of selected autochthonous soil bacteria for the removal of aromatic hydrocarbons from a heavily polluted soil. This result disagrees with that found by Lehtomaki et al. (1975). They reported that oil-degrading bacteria added to samples of soil contaminated with light fuel oil or heavy waste oil had no significant effect on the oil degradation. The difference can be attributed to the higher hydrocarbon concentration added in our experiment, 10% as compared with 0-5% in Lehtomaki's work. High hydrocarbon concentration and thorough mixing seem to be very important factors to take into account when an inoculation program is planned. As has been suggested by Goldstein et al. (1985) a low concentration of the inoculant in nature does not support the growth of the inoculated species, or it would be slower than the rate of predation, so that the activity of the grazers will reduce the cell density of the added organisms. A good degree of mixing is necessary to assure the contact of the added organisms with the pollutants to be destroyed.

Inoculant production Two isolates were identified as members of the genus Pseudomonas and the third is tentatively classified as an Alcaligenes sp. All of them were capable of utilizing the aromatic hydrocarbon mixture as the sole carbon source. For the three bacterial strains, growth at room temperature on nutrient supplemented vermiculite produced final products with population densities o f 109 to 101° in 21 days (Table 2). The ability of utilizing hydrocarbons for growth may be a variable character and easily lost during cultivation in hydrocarbon free media

G. L Vecchioli, M. T. Del Panno, M. T. Painceira


TABLE 2 Production of Inoculants Determined in PC Agar


Number of baeteria/g Initial inoculum

After 21 days

After 60 days

Pseudomonas sp FN775-1 FN775-5

5'5 10 6

3"0 10 9

3'4 107

1"7 10 9

1"3 10 6 1'3 108

1'1 l0 T

7'7 10 9

2'0 105

Alcaligenes sp FN775-2

(Jensen, 1975). The capability of the isolated strains to preserve this ability when grown in supplemented vermiculite was determined by enumeration in SMM with hydrocarbons as sole source of carbon. Table 3 shows the stability of Pseudomonas FN775-5 inoculant stored at room temperature for at least 2 months. Pseudomonas FN775-1 inoculant is stable for a month. In both cases their hydrocarbon degrading capabilities are fully preserved. The Alcaligenes strain degrading capability remains in the values of inoculation after 21 days. In this paper we demonstrate that although hydrocarbon degrading bacteria are naturally present in fertile soils, microbial inoculation is capable of substantial acceleration of biodegradation whenever appropriate conditions are provided. The strains used preserved their hydrocarbon degrading capability for at least a month when grown in hydrocarbon free nutrient supplemented vermiculite and required no special storage. We conclude that biodegradation of heavily polluted soils such as those found in land treatment facilities or accidental spills may be enhanced by inoculation with selected indigenous TABLE 3 Production of Inoculants Determined in SMM


Number of bacteria/g Initial inoculum

After 21 days

After 60 days

5'3 10 6

1"6 107

3"0 10 9 2"4 108

2"0 104 1'2 108

1-0 107

1-6 107

7.5 104

Pseudomonas sp FN775-1 FN775-5

Alcaligenes sp FN775-2

Enhancing degradation qf hydrocarbons in soil


bacteria and that a support such as vermiculite can be used for easy inoculant production.

A C K N O W L E D G E M ENTS This work was supported by Petroquimica General Mosconi SAIyC (PGM) and Secretaria de Ciencia y T6cnica (SECYT). We thank Graciela Pietracatella and R o b e r t o More of P G M ' s L a b o r a t o r y for analytical determinations and Marta Ruiz for technical assistance.

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G. L Vecchioli, M. T. Del Panno, M. T. Painceira

Painceira, M. T. & Molo, M. (1988). A Pseudomonas using hydrocarbons obtained by genetic techniques. Microbios Letters, 39, 33-6. Solari, A. A. & Painceira, M. T. (1976). Estudio de una Pseudomonas aislada del suelo que es capaz de utilizar el naftaleno como fuente de carbono. Acta Biog. Clin. Lat., 10, 321-6. Torstensson, L. (1988). Microbial decomposition of herbicides in the soil. Outlook in Agriculture, 17, 120~4. Vecchioli, G. I., Succar, S. D., Molo, M., Carri, M., Del Panno, M. T. & Painceira, M. T. (1988). Degradation of hydrocarbons contained in bottom sludge and flocculated material floating in API basins. (In Spanish). Ind. Quim., 289, 29-32. Walter, M. V., Barbour, K., McDowell, M. & Seidler, R. J. (1987). A method to evaluate survival of genetically engineered bacteria in soil extracts. Current Microbiology, 15, 193-7.