effect of photosynthetic bacteria and compost on degradation of petroleum products in soil

0003-6838/04/4002- © 2004

MAIK “Nauka

/Interperiodica”0181

Applied Biochemistry and Microbiology, Vol. 40, No. 2, 2004, pp. 181–185. Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 40, No. 2, 2004, pp. 214–219.Original Russian Text Copyright © 2004 by Ten, Kirienko, Imranova.

The literature contains a large body of evidence onthe correlation between the microbial complex and oilpollution of soil [1, 2]. Practical recommendations forland reclamation include application of organic fertiliz-ers [1, 3] and planting of grasses [1, 4]. On the otherhand, there is evidence that photosynthetic bacteriamay affect the process of degradation of petroleumproducts in an aquatic medium. In addition, photosyn-thetic bacteria were found to grow symbiotically withorganotrophic microorganisms causing degradation ofhigh-molecular-weight organic substances [5].

The goal of this work was to study the effect of com-post and liquid culture of photosynthetic bacteria ongrowth of hydrocarbon-oxidizing microorganisms andto test the possibility of the use of photosynthetic bac-teria for bioremediation of soil polluted with petroleumproducts.

MATERIALS AND METHODS

Scheme of model experiments.

In the control variantof the experiment, 65 g of diesel fuel (DF) was added toa 500-g sample of arable soil, which corresponded to alevel of pollution of a soil surface layer of 10 l/m

2

. Inanother variant of the experiment, 10 ml of a suspen-sion of the photosynthetic bacteria (PSB)

Rhodobactercapsulata

and

Rhodocyclus gelatinosa

, containing

10

9

–10

10

cells per ml, were also added to the same amountof soil (equivalent to 1.5 l/m

2

of soil surface). The cul-tures of these species of photosynthetic bacteria wereisolated from commercial preparations available fromJapan and stored in the Laboratory of Microbiology,Institute of Aquatic and Ecological Problems, Far EastDivision of the Russian Academy of Sciences. In thethird variant of the experiment, 27 g of wood compostwas added to 500 g of polluted soil (equivalent to40 t/ha). In the fourth variant of the experiment, similar

samples of liquid culture of photosynthetic bacteria andcompost were simultaneously added to polluted soil.

All soil samples were kept in glass beakers at

27°ë

(thermostated) for 10 days.The effect of hydrocarbon-oxidizing and photosyn-

thetic bacteria on degradation of diesel fuel was studiedin a liquid medium of the following composition (g/l):

KH

2

PO

4

, 0.8; NH

4

Cl, 0.5; NaHCO

3

, 0.5; MgSO

4

, 0.2;CaCl

2

, 0.05; and yeast extract, 0.1; diesel fuel wasadded at a ratio of 0.5 ml per 1 l. In one of the experi-mental variants, a suspension of hydrocarbon-oxidizingmicroorganisms preisolated from soil polluted withdiesel fuel was added to 1 l liquid medium. In anothervariant of the experiment, a sample of 50 ml of liquidculture medium containing two species of photosyn-thetic bacteria was also added to the system. After inoc-ulation of the microorganisms, the experimentalmedium was aerated in a 2-l flask using a portable com-pressor. The rate of air flow was 500 ml/min. The flaskswere illuminated with a common incandescent 60-Wlamp and kept at a temperature ranging from 25 to

30°ë

. The microorganisms were cultivated for 10 days.

Scheme of field experiments.

In the field experiment,waste engine oil (a mixture of waste petroleum prod-ucts, MWPP) was added instead of diesel fuel. In con-trast to diesel fuel, MWPP contained less light fractionsand more heavy fractions.

A

5

×

5

m plot of inundation meadow at the AmurRiver bank was treated with 10 l of the pollutant (equiv-alent to 130 ml/kg soil). The pollutant was uniformlyspread over the soil surface. In one variant, the sameplot was treated with a liquid culture of photosyntheticbacteria, containing

10

9

–10

10

cells/ml, in a dose of1.5 l/m

2

. To provide for uniform spread of the PSB cul-ture over the soil surface, the culture was diluted five-fold with water before the treatment. In another variant,wood compost was added to soil together with PSB

Effect of Photosynthetic Bacteria and Compost on Degradation of Petroleum Products in Soil

Kh. Ten, O. A. Kirienko, and E. L. Imranova

Institute of Aquatic and Ecological Problems, Far East Division, Russian Academy of Sciences, Khabarovsk, 680000 Russiae-mail: [email protected]

Received March 17, 2003

Abstract

—Addition of diesel fuel and waste engine oil to soil was found to stimulate hydrocarbon-oxidizingmicroorganisms. Corynebacteria constitute a large group of hydrocarbon-oxidizing microorganisms. Additionof a liquid culture of photosynthetic bacteria to soil facilitates degradation of petroleum products and also stim-ulates growth of hydrocarbon-oxidizing microorganisms. Combined addition of photosynthetic bacteria andcompost to soil polluted with petroleum products produces a greater increase in the number of hydrocarbon-oxidizing bacteria and substantially augments the rate of pollutant degradation.

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et al

.

culture. The dose of wood compost in this case wasequal to that in the model experiment described above.

Microbiological analysis of soil.

The total count ofmicroorganisms was determined by the method of five-fold plating of soil suspension in the following agarmedia: beef–peptone agar (BPA) diluted 1 : 10 (forcounting bacterial cells), starch–ammonia agar (forcounting actinomycetes), and acidified Czapek medium(for counting fungi) [6].

Hydrocarbon-oxidizing bacteria were grown on anagar medium, the mineral composition of which wasdescribed above (model experiment). Waste engine oilwas added to culture medium (5 ml/l culture medium)after the medium had been sterilized.

It was shown in preliminary experiments that stor-age of waste engine oil was accompanied by its con-tamination with microorganisms. Therefore, the wasteengine oil samples intended for experimental studywere sterilized by five cycles of treatment in a micro-wave oven (2 min each cycle; microwave power,900 W). After the nutrient medium with waste engineoil had been thoroughly shaken and poured into petri

dishes, the oil droplets appearing on the agar surfacewere uniformly spread with a spatula.

Content of petroleum products.

The content of die-sel fuel in liquid medium was determined by extractionwith chloroform (1 : 1, vol/vol) followed by evapora-tion, additional drying, and gravimetric determinationof hydrocarbons [7]. The total biomass carbon was alsodetermined in the culture medium, by the method ofoxidation of hydrocarbons using the chromium mixturedescribed in [8]. Liquid culture was prefiltered througha membrane filter to remove microbial cells.

The content of waste engine oil in soil was deter-mined as described in [9]. This method is based onextraction of petroleum products with carbon tetrachlo-ride, filtration through a column packed with an alumi-num sorbent, and evaporation of the solvent in a weigh-ing bottle (under airflow; room temperature) to a con-stant weight of the residue (which is recorded). Theenzyme activity was assayed as described in [10].

RESULTS AND DISCUSSION

There was a 10% decrease in the initial content ofdiesel fuel in soil 30 days after pollution (Table 1).Addition of compost caused a 2.5-fold increase in therate of degradation of diesel fuel. One month after addi-tion of a liquid culture of photosynthetic bacteria, thesoil contained 50% of the initial concentration of petro-leum products. More than 70% of the initial content ofDF degraded in the variant with compost and liquid cul-ture of PSB.

It was shown in experiments with liquid mineralmedium that addition of hydrocarbon-oxidizing bacte-ria was accompanied by a time-dependent decrease inthe content of both total carbon and DF in the medium.Addition of a liquid culture of photosynthetic bacteriafurther increased the rate of degradation of petroleumproducts (Table 2).

Table 1.

Content of diesel fuel in soil after 30 days of themodel experiment*

Variantof experiment DF, g/kg soil DF, %

Initial 130.0

±

20 100.0

Control 115.8

±

12 89.1

Compost 97.5

±

08 75.0

PSB 66.4

±

06 51.1

PSB + compost 38.1

±

08 29.3

* (DF) diesel fuel; (PSB) photosynthetic bacteria.

Table 2.

Changes in the content of diesel fuel in the model experiment with liquid medium*

Duration of experiment, daysTotal content of C, % Content of DF, g/l medium

HOB HOB + PSB HOB HOB + PSB

Initial 0.57 (100) 0.57 (100) 5.00 (100) 5.00 (100)

1 0.45 (78.9) 0.39 (68.4) 4.30 (86) 3.80 (76)

5 0.41 (71.9) 0.21 (36.8) 3.75 (75) 3.07 (61.4)

9 0.35 (61.4) 0.15 (26.3) 3.60 (72) 2.34 (46.8)

13 0.28 (49.1) 0.09 (15.8) 3.07 (61.4) 1.30 (26)

18 0.23 (40.3) 0.08 (14.0) 2.58 (51.6) 0.70 (14)

* (HOB) hydrocarbon-oxidizing bacteria; (PSB) photosynthetic bacteria; residual content (% of initial level) is given in parentheses.


Vol. 40

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EFFECT OF PHOTOSYNTHETIC BACTERIA 183

Artificial pollution of meadow soil with MWPPcaused an abrupt decrease in the total count of bacteriaand fungi and complete extinction of actinomycetes(Figs. 1 and 2). As the time passed, there was a gradualrestoration of the general microbial cenosis, although ata quantitatively lower level. Growth of bacteria con-taining pink, orange, and yellow pigments predomi-nated. In accordance with the classification given in[11], morphological and physiological characteristicsof many dominant bacterial strains were close to thoseof the genera

Rhodococcus

,

Arthrobacter

, and

Brevi-bacterium.

It was reported [12, 13] that petroleumproducts initiated growth of hydrocarbon-oxidizingbacteria (particularly those of the genus

Rhodococcus

).It was found that treatment of polluted soil with a liq-

uid culture of photosynthetic bacteria stimulated growthof the bacterial population, whereas combined additionof photosynthetic bacteria and compost caused a sharpincrease in the count of microorganisms (Fig. 3).

It should be noted that petroleum pollution causedelimination of actinomycetes and many fungi, whichwas paralleled by a significant decrease in the totalcount of many species of microorganisms. On the otherhand, in all experimental variants with petroleum pollu-tion, there was an increase in the count of representa-tives of such fungal genera as

Trichoderma

,

Penicil-lium

, and

Mucor

. The fact of conservation of thesefungi in the resistance zone under the conditions ofsevere petroleum pollution was also reported by Zvyag-intsev

et al.

[14]. According to the results reported in[14], the amount of waste engine oil added to soil in ourexperiments (130 ml/kg soil) corresponded to a high-level pollution.

Treatment of polluted soil by combined addition ofphotosynthetic bacteria and compost resulted in theappearance of several species of actinomycetes and inan insignificant growth of the fungal community. Theinitial level of population density was approached inone year.

The content of waste engine oil in soil decreased inaccordance with increases in the counts of microorgan-isms (particularly hydrocarbon-oxidizing bacteria)(Table 3). In control variants of soil pollution withpetroleum products, about 30% of the initial amount ofthe pollutant underwent degradation within one year.Addition of a liquid culture of photosynthetic bacteriato polluted soil significantly increased the rate of deg-radation of MWPP, and the residual content of the pol-lutant retained in the soil after one year was 30% belowits initial level. Biodegradation of petroleum productswas still more substantial in the case of treatment ofpolluted soil with a combination of compost and photo-synthetic bacteria. Under conditions of such treatment,about 85% of the initial amount of pollutant wasdegraded within one year.

It follows from Table 4 that soil pollution with wasteengine oil caused a significant increase in soil acidity,which was accounted for by production and accumula-

50

1

0

2 3 4 1 2 3 4 1 2 3 4

(‡)

(b)(c)

100150200250300350400450

million per gram

Fig. 1.

Changes in the count (million cells per gram) of bac-teria in soil: (

1

) control (without pollution); (

2

) after pollu-tion with petroleum products; (

3

) after addition of PSB;(

4

) after addition of PSB + compost; (a) 7 days after pollu-tion; (b) 12 months after; (c) averaged over five assays.

Fig. 2.

Changes in the count (thousand specimens per gram soil)of fungi induced by soil treatment. Designations as in Fig. 1.

50

1

0

2 3 4 1 2 3 4 1 2 3 4

(‡)

(b)(c)

100

150

200

250

300

thousand per gram

Fig. 3.

Growth of bacterial colonies in suspension of soilpolluted with petroleum products (micrograph): (

1

) control;(

2

) after treatment with PSB; (

3

) the same + compost.

1

2 3

184


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tion of organic acids. In the case of soil treatment witha preparation of photosynthetic bacteria, there was adecrease in soil acidity and an insignificant increase inthe concentrations of carbon and nitrogen. More signif-icant changes in the properties of soil (reduced acidityof soil extract and increased total content of carbon andnitrogen) were observed in the case of combined addi-tion of compost and photosynthetic bacteria. This wasalso accompanied by activation of peroxidase andpolyphenol oxidase, which were responsible for degra-dation of aromatic compounds and their condensationand transformation to humic substances. As for suchenzymes as invertase, catalase, and dehydrogenase, wedid not find any statistically significant difference

between their activities in control and polluted variantsof soil. A similar lack of effect of petroleum pollutionon their activity was reported in [15, 16].

Many species of photosynthetic bacteria have nitro-gen-fixation activity in symbiosis with organotrophicmicroorganisms. These microorganisms use low-molecular-weight organic compounds (e.g., fatty acids)as donors of hydrogen [5, 17]. Processes of degradationof organic substances in soil polluted with petroleumproducts are accompanied by considerable uptake ofoxygen and inhibition of activity of hydrocarbon-oxi-dizing bacteria. These effects are thought to be associ-ated with accumulation of butyric acid produced underanaerobic conditions [13].

Treatment of soil characterized by a similar degreeof pollution with a liquid culture of photosynthetic bac-teria facilitates degradation of petroleum products. Per-haps, this can be explained by the consumption ofbutyric acid and other low-molecular-weight organicacids as sources of carbon nutrition, resulting in favor-able conditions for the vital activity of hydrocarbon-oxidizing bacteria. On the other hand, it is well knownthat many species of photosynthetic bacteria are capa-ble of degrading low-molecular-weight organic aro-matic compounds contained in petroleum products orsubstances produced as a result of degradation of petro-leum products [18].

Table 3.

Content of waste engine oil (MWPP) in soil after 1year

Variant of experiment MWPP,g/kg soil MWPP, %

Control (addition of 10 l/m

2

) 130

±

25 100.0

Control 87

±

32 66.9

Treatment with PSB 36

±

18 27.7

Treatment with PSB + compost 19

±

12 14.6

Table 4.

Changes in chemical properties and enzymatic activity of soil polluted with petroleum products

Variant of experiment pH Total C, % Total N, % Peroxidase* Polyphenol oxidase*

Control (without pollution) after 10 days:

without treatment 6.06 4.09 0.20 48.0 30.0

treatment with PSB 6.10 4.54 0.53 49.5 42.0

treatment with PSB + compost 6.32 5.56 0.50 54.0 69.0

Control (without pollution) after 6 months:




10 days after pollution:




6 months after pollution:




* 1 mg galline purple per gram soil.


Vol. 40

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EFFECT OF PHOTOSYNTHETIC BACTERIA 185

The addition of compost to soil enriches it withorganic compounds and a whole microbial association,including various species of hydrocarbon-oxidizingbacteria and other heterotrophic microorganisms. Thelow-molecular-weight organic acids produced underconditions of oxygen deficiency acidify the soil solu-tion and serve as sources of carbon nutrition for photo-synthetic bacteria, as evidenced by the results discussedabove. These facts can explain the stimulatory effect onprocesses of petroleum product degradation observedafter additional soil treatment with a culture of photo-synthetic bacteria.

Certain species of photosynthetic bacteria are usedsuccessfully for purification of wastewater enrichedwith organic compounds. The wastewater treated bythis method and slime containing cells of these micro-organisms in large amounts are used in many areas ofenvironment preservation [5, 18, 19].

The experimental results described in this work pro-vide evidence that treatment of soil with compost and aliquid culture of photosynthetic bacteria exerts a signif-icant effect on the biodegradation of petroleum prod-ucts. These findings are of considerable practicalimportance for reclamation of lands polluted with oil.

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effect of photosynthetic bacteria and compost on degradation of petroleum products in soil

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