Appl Microbiol Biotechnol (1986) 25:295--299 Applied Microbiology

Biotechnology © Springer-Verlag 1986

Changes in the bacterial community after application of oily sludge to soil

Aaslaug Lode

Department of Microbiology, Agricultural University of Norway, Box 40, N 1432 Aas-NLH, Norway

Summary. The changes in the bacterial flora after application of oily sludge and fertilizer to two dif- ferent soils, both sandy, but one somewhat richer in organic material, have been studied. Applica- tion of oily sludge and fertilizer to the soils had no influence on direct counts. The CFUs in- creased in the low-organic soil when sludge was applied, but did not change in the richer soil by the same treatment. When both sludge and ferti- lizer were applied together, strong increases in CFUs were found in both soils. Application of fertilizer together with sludge excluded spore- forming rods and actinomycetes from the poorer soil and strongly stimulated pleomorphic rods + cocci and non-sporeforming regular rods. The same treatment of the richer soil resulted in an enhancement of CFUs of all tested groups.

Introduction

Landfarming is a well-known and recognized method to obtain biodegradation of oily wastes (Dotsen et al. 1971; Jensen 1974; Cansfield and Racz 1978). The practical application of the method requires an enhanced biodegradation process. This process depends on the oil composi- tion, the nature of the microbial community, and on variations of the environmental factors. By knowing the part of the microorganisms being most active in degrading hydrocarbons under the given circumstances, the basis for obtaining an enrichment of hydrocarbon degrading microor- ganisms in soil is obtained.

Offprint requests to: A. Lode

Prior to a possible large scale use in South- Norway, experiments were performed on small field plots to explore how the process functioned under local conditions (Sandvik et al. 1986). In this paper, a detailed report on qualitative changes in the bacterial community of the tested soils, is given.

Materials and methods

Two experimental fields were chosen. The sites of the fields, the properties of the soils and oily sludge, and the soil and sludge treatment are described elsewhere (Sandvik et al. 1986). The soil samples were collected as earlier described, but in this case the samples were subjected to microbial analyses immedi- ately after collection.

For each soil sample two parallel microbial analyses were performed. The total bacterial numbers were determined as di- rect counts using fluorescence microscopy. The soil suspen- sions were stained with acridine orange, and the organisms collected on Nuclepore filters pre-treated with irgalan black (Hobbie et al. 1977). Three filters were counted for each sus- pension. Colony forming units (CFUs) were determined on soil extract agar supplemented with 40 ppm cycloheximide (Sigma) (Holm and Jensen 1972). Counting was done under a binocular microscope after four weeks of incubation at 21°C.

For a rough classification of the bacteria, plates contain- ing between 50 and 100 colonies were chosen. All colonies from each of these plates were transferred to slants of soil ex- tract agar. For each soil sample, 250 to 300 isolates were stud- ied. The following morphological characters were observed after incubation at 21 ° C for 18--48 h: shape and arrangement of the cells, presence of endospores, pigmentation, and Gram reaction. According to these characters the bacteria were clas- sified in one of 6 groups.

Results

The effects of application of oily sludge and fer- tilizer on the direct counts and CFUs of bacteria in the two tested soils, are shown in Table 1.

296 A. Lode: Soil bacteria and oily sludge

Table 1. Influence of oily sludge and nitrogen fertilizer on direct counts and on colony forming units (CFU) (both in million per g dry soil) of bacteria 3 months after application

Square 1 Square 1

pH Moisture Direct CFU CFU pH Moisture Direct CFU CFU content, counts (D) --D---' 100 content, counts (D) ~ - - . 100 % of dry m. % of dry m.

Field 1 SoNo 5.2 6.1 2300 9 0.4 5.1 2.9 2400 5 0.2 S~No 6.7 10.7 2400 321 13.4 6.9 5.1 1900 75 3.9 SoN6o0 5.6 5.4 2600 17 0.7 5.4 3.2 2700 13 0.5 SIN6oo 6.7 7.6 2100 1070 51.0 6.8 5.2 2300 247 10,7

Field 2 SoNo 4.9 14.9 3800 64 1.7 4.9 11.6 3700 59 1.6 SlNo 6.2 12.6 4000 47 1.2 6.3 11.6 4400 87 2.0 SoN6o0 4.8 16.0 4600 74 1.5 5.2 10.6 3700 36 1.0 S1N6oo 6.4 13.4 3800 284 7.5 6.4 10.3 4200 930 22.1

Field 1 : Low in organic material. Field 2: Higher in organic material. Square 1 and 2: Two parallel parts of the same field.

Abbreviations: So: control soil No: no nitrogen applied $1: oily sludge applied N6oo: 600 kg N / h a applied

Table 2. Percentage distribution of CFUs between different bacterial groups in soil samples 3 months after sludge application

Field 1 Field 2

Groups SoNo S1 No So N600 S1N600 So No S 1 No SoN6oo S I N6oo

1. Sporeforming rods 10 2 9 0 19 3 6 1 2. Actinomycetes, mainly Streptomyces 25 0 13 0 9 4 14 2 3. Pleomorphic rods + cocci 31 18 20 25 28 22 25 17 4. Non-sporeforming regular rods.

Gram + 7 13 10 7 11 11 6 13 5. Nou-sporeforming regular rods.

G r a m - 19 67 41 66 23 55 38 59 (Without pigments) (9) (28) (11) (19) (11) (11) (17) (24) (Pigmented) (10) (39) (30) (47) (12) (44) (21) (35)

6. No growth after transferring 8 0 7 2 10 5 11 8

Total 100 100 100 100 100 100 100 100

Explanations: see Table 1

No significant changes in the direct counts of bacteria could be observed after application of oily sludge or fertilizer to the soil. However, the direct counts of bacteria were about 70% higher in field 2 than in field 1.

Nevertheless, the applications of oily sludge and fertilizer to the soil strongly influenced the number of CFUs. In soil from untreated plots of field 1, CFUs represented 0.2 and 0.4% of the di- rect bacterial counts. This percentage increased to 3.9 and 13.4% with addition of sludge alone, and to a respective 10.7 and 51% in the two squares when both sludge and fertilizer were applied. Ap- plication of fertilizer alone seemed to have mini- mum effect on the active microflora in this field.

In the control areas in field 2, CFUs were 1.6 and 1.7% of the direct counts. Separate applica- tion of fertilizer or oily sludge seemed not to in- fluence the CFU. When both fertilizer and oily sludge were applied to the same plots, a distinct increase in C FU was observed also in this field.

The soil pH showed a significant increase in the oil treated plots (Table 1). In field 1 the mois- ture content was higher in plots treated with sludge than in the controls. No corresponding ef- fect was found in field 2.

The results of the rough morphological classi- fication are presented in Tables 2, 3 and 4. In the control plots of field 1, a predominance of actino- mycetes, pleomorphic rods + cocci and Gram-,

A. Lode: Soil bacteria and oily sludge 297

Table 3. CFUs (million per g dry soil) of bacteria in different morphological groups in soil samples fromfield I

Groups

Square 1 Square 2

SoNo SlNo SoN6oo S1N6oo SONO SINO SoN60o &Nmo - - --

1. Sporeforming rods 0.9 6.4 1.5 - 0.5 1.5 1.2 -

2. Actinomycetes, mainly Streptomyces 2.2 - 2.2 - 1.2 - 1.7 -

3. Pleomorphic rods + cocci 2.8 57.7 3.4 267.5 1.6 13.5 2.6 61.8 4. Non-sporeforming regular rods.

Gram + 0.6 41.7 1.7 74.9 0.3 9.8 1.3 17.3 5. Non-sporeforming regular rods.

Gram - 1.7 215.1 7.0 706.2 0.9 50.2 5.3 163.0 (Without pigments) (0.8) (89.9) (1.9) (203.3) (0.4) (21.0) (1.4) (46.9) (Pigmented) (0.9) (125.2) (5.1) (502.9) (0.5) (29.2) (3.9) (116.1)

6. No growth after transferring 0.7 - 1.2 21.4 0.4 - 0.9 4.9

Total 8.9 320.9 17.0 1070.0 4.9 75.0 13.0 247.0

Explanations: see Table 1

Table 4. CFUs (million per g dry soil) of bacteria in different morphological groups in soil samples fromfield 2

Square 1 Square 2

Groups SoNo &NO SoN60o S I N ~ O O SONO SINO SoN60o SlN600

1. Sporeforming rods 12.2 1.4 4.4 2.8 11.2 2.6 2.1 9.3 2. Actinomycetes, mainly Streptomyces 5.8 1.9 10.4 5.7 5.3 3.5 5.0 18.6 3. Pleomorphic rods + cocci 17.9 10.3 18.5 48.3 16.5 19.1 9.0 158.1 4. Non-sporeforming regular rods.

Gram + 7.0 5.2 4.4 36.9 6.5 9.6 2.2 120.9 5. Non-sporeforming regular rods.

Gram - 14.7 25.9 28.1 167.6 13.6 47.9 13.7 548.7 (Without pigments) (7.0) (5.2) (12.6) (68.2) (6.5) (9.6) (6.1) (223.2) (Pigmented) (7.7) (20.7) (15.5) (99.4) (7.1) (38.3) (7.6) (325.5)

6. No growth after transferring 6.4 2.4 8.1 22.7 5.9 4.4 4.0 74.4

Total 64.0 47.1

Explanations: see Table 1

non-sporeforming regular rods was observed. In the corresponding soil from field 2, sporeformers together with the pleomorphic rods + cocci and Gram-, non-sporeforming regular rods seemed to be in higher proportions (Table 2). The distribu- tion of bacteria between the other groups was uni- form, varying between 7 and 10% in samples from field 1, and between 9 and 11% in samples from field 2.

Application of oily sludge to the soil changed the composition of the microflora in both fields (Table 2). In field 1 the sporeformers decreased in relative, but increased in absolute numbers when sludge alone was added (Table 3). When the sludge treated soil was fertilized, these bacteria seemed to be totally excluded (Table 2). Applica- tion of oily sludge to the soil in Field 2 reduced

both the relative and the actual numbers of spore- forming bacteria per g of soil, independent of fer- tilization (Table 4).

Actinomycetes could not be isolated from sludge-treated plots in field 1 (Table 2). The rela- tive content of these bacteria was very low also in the corresponding soil samples from field 2. Al- though application of sludge reduced the number of actinomycetes, a simultaneous application of fertilizer to this soil seemed to neutralize the inhi- bitory effect of sludge (Table 4).

The relative contents of pleomorphic rods + cocci, and Gram + , non-sporeforming regular rods decreased or showed minimum changes in soil treated with oily sludge in field 1 (Table 2). Nevertheless, considerably higher numbers of these bacteria were found in the treated soil than

298

in the controls (Table 3). The enhancements were strongly stimulated by application of fertilizer. In field 2 addition of sludge alone seemed not to give a corresponding positive effect on these bac- teria as in field 1. Nevertheless, when sludge treated soil was fertilized, the number of pleo- morphic rods + cocci, and Gram+, non-spore- forming rods increased considerably also in this soil.

G r a m - , regular rods formed the only group which showed a distinct increase in relative num- bers after application of oily sludge and/or ferti- lizer (Table 2). In field 1, this group represented two of three bacteria in sludge treated soil, in field 2 a somewhat lesser proportion, but still more than 50%. It should be noted that particu- larly the pigmented types within this bacterial group seemed to increase in numbers when sludge was applied.

Discussion

After application of oily sludge to the soils, varia- tions in the values of CFUs between correspond- ing squares were observed (Table 1). The reasons are especially connected with the inhomogeneity of the sludge and with the practical problem in obtaining a uniform distribution of the sludge on the soil surface (Sandvik et al. 1986). However, CFUs increased when oily sludge was applied to soil (Table 1). Fertilizing further enhanced the stimulating effect. It seems to be a generally oc- curring phenomenon that application of certain amounts of hydrocarbon containing material to soil increases viable counts and stimulate micro- bial activity (Jobson et al. 1974; Jensen 1975; At- las 1981). The effect has been observed in temper- ate and arctic climates (Dotson et al. 197l; Sex- tone and Atlas 1977) and with different kinds of oil and oily wastes (Jensen 1974; Raymond et al. 1976; Watkinson 1978).

The positive effect of application of oily sludge and fertilizer on CFUs seemed to be higher in the soil with the lowest content of organic ma- terial (field 1). Nevertheless, Sandvik et al. (1986) found, during the first months, that the reduction of oil content in soil for all treatments was greater in field 2 than in field 1. This observation sup- ports the assumption that cooxidation of hydro- carbons (Perry 1979), together with increased wa- ter capacity (Table 1), favour oil degradation in soil.

Application of oily sludge to the soil seemed to have no influence on direct counts of bacteria.

A. Lode: Soil bacteria and oily sludge

In all the controls and in the majority of the treated plots, CFU represented only a small part of the direct counts. Consequently, a change in CFUs would hardly influence the direct counts. It is remarkable that even the highest increase in CFUs in one of the sludge and fertilizer treated plots in field 1 (CFU 51% of total number) was not observed as a simultaneous increase of the di- rect counts. This might indicate that many bacte- ria in soil occur in a state of dormancy waiting for a suitable material for reactivation (Alexander 1977). A part of this bacteria might be hydrocar- bon degraders, but many of them are likely to be organisms living on intermediary degradation products.

In Table 5, the ratios between CFUs in oil treated soils and the corresponding control soils are shown. After sludge application, CFUs in- creased within the groups 1, 3, 4, and 5 in field 1, but only within the groups 3, 4 and 5 in field 2 (SINo/SoNo). Since the oil degradation was high- est in field 2 during the first few months after sludge applications, the observation indicates that the most active hydrocarbon degraders occurred within the last three groups. This observation is in accordance with results earlier reported in the lit- erature. Pleomorphic rods, including Corynebac- terium, Arthrobacter and Nocardia are often re- ported as utilizers of hydrocarbons. The same is true for bacteria belonging to Pseudomonas (Jones and Edington 1968; Kincannon 1972; Jensen 1975), whereas sporeforming rods together with actinomycetes seem to show little or no positive response on hydrocarbon application to soil (Kra- silnikov et al. 1969; Dotson et al. 1971; Kincan- non 1972; Jensen 1975). Some sporeformers have certainly shown the ability to oxidize alkanes in the presence of other carbon sources (Kachholz and Rehm 1977), and some Streptomyces strains, able to utilize hydrocarbons, have been isolated from oil-treated soil (Krasilnikov et al. 1969).

By comparing the ratios in the columns S1No/ SoNo and SiN6oo/SoN6oo, the effect of oily sludge addition on the different bacterial groups in non- fertilized and fertilized soils could be studied. If the attention is focused on groups 3, 4 and 5 in field 1, lower ratios are observed for the groups 4 and 5 in soil treated with fertilizer than in the cor- responding control soil, but considerably higher ones for group 3. The stimulation of the oil de- grading bacteria in the pleomorphic group in re- sponse to adding fertilizer may indicate that pleo- morphic bacteria adapt to oil degradation more easily than others under improved growth condi- tions. The very high stimulation of non-spore-

A. Lode: Soil bacteria and oily sludge

Table 5. The ratio between CFUs in oil treated soils and control soils i n f i e l d 1 and 2

299

Field 1 (Square 1) Field 2 (Square 2)

Groups S1No/SoNo S1N6oo/SoNo S1Nroo/SoN6oo S1No/SoNo SiN6oo/SoNo S1N6oo/SoN6oo

1. Sporeforming rods 7.1 -- -- 0.2 0.8 4,4

2. Actinomycetes, mainly S t rep to - myces - - - - 0.7 3.5 3.7

3. Pleomorphic rods + cocci 20.6 95.5 78.7 1.2 9.6 17.6

4. Non-sporeform- ing regular rods. Gram+ 69.5 124.8 44.1 1.5 18.6 55.0

5. Non-sporeform- ing regular rods. Gram- 126.5 415.4 100.8 3.5 40.3 40.1 (Without pig- ments) (112.4) (254.1) (107.0) (1.5) (34.3) (36.6) (Pigmented) (139.1) (558.8) 98.6) (5.4) (45.8) (42.8)

6. No growth after transferring -- 30.6 17.8 0.7 12.6 18.6

Explanations: see Table 1

forming regular rods after sludge and fertilizer a p p l i c a t i o n (S1N6oo/SoNo) supports the assump- tion that many of these rods (particularly the pig- mented types) live on degradation products of hy- drocarbons.

The enhancement of bacteria within all the tested groups, after application of oily sludge to fertilized soil in field 2 (S1N6oo/SoN6oo), may be explained by the higher content of organic mate- rial in this soil.

A c k n o w l e d g e m e n t . I thank Tor Arve Pedersen for excellent support in the preparation of this manuscript.

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Received June 2, 1986/Revised August 25, 1986