Potential for remediation of water repellent soils by ... - CiteSeerX

S358

Biologia, Bratislava, 61/Suppl. 19: S358—S362, 2006

Potential for remediation of water repellent soils by inoculation with wax-degrading bacteria in south-western Australia Margaret M. Roper CSIRO Plant Industry, Private Bag No. 5, Wembley, WA 6913, Australia; tel.: +61-8-9333 6668, Fax: +61-9387-8991, e-mail: [email protected]

Abstract: Water repellency resulting from waxy coatings around soil particles causes significant crop and pasture losses. Bioremediation of these soils using inoculation of wax-degrading bacteria was investigated under field conditions. In a small scale experiment without any additional nutrients or soil conditioners, 2 inoculants (Rhodococcus sp. and Roseomonas sp.) out of 7 resulted in significant improvements in water infiltration. A larger scale experiment had compost and fertiliser applied to support inoculants in nutrient-poor sands and lime was added to half the treatments. There were 6 different inoculants and their mixtures. One inoculant (Mycobacterium sp.) significantly reduced water repellency on its own. However, the addition of lime produced a significant “inoculant by lime” interaction, and limed treatments with each of the 5 individual cultures of Rhodococcus spp. and a mixture containing the 5 cultures of Rhodococcus spp. and 1 culture of Mycobacterium sp. all resulted in significant reductions in water repellency when compared with their non-limed counterparts and controls. Lime alone (1 t/ha, 70% neutralising value) produced a small but significant benefit compared with the non-limed control. The results indicate that there is potential to increase soil wettability through increased activity by wax-degrading bacteria. Key words: actinomycetes, bacteria, hydrophobicity, MED, repellency, wax degradation

Introduction Water repellency, which affects > 5 million hectares of western and southern Australia, is mostly associated with siliceous sands (Tate et al., 1989). Repellency causes uneven infiltration of water into soil (Ritsema et al., 1997) and this reduces crop and pasture yields through uneven and delayed germination, poor stand establishment, and increased risk of erosion from wind and water (Bond, 1964; King, 1981; Tate et al., 1989). Water repellency is primarily caused by a skin around soil particles of waxes consisting of branched and unbranched C16 to C36 fatty acids, and their esters, alkanes, alcohols, and sterols (Roberts & Carbon, 1972; Ma’shum et al., 1988; Franco et al., 1995, 2000; Morley et al., 2005). Kaolinite has been used successfully to ameliorate water repellency (Ma’shum et al., 1989; Blackwell, 1993; Ward & Oades, 1993; McKissock et al., 2000, 2002; Lichner et al., 2006), but large amounts (100 t/ha) are required (Blackwell, 1993) and therefore this strategy is only economic if clay occurs on site. Wetting agents (surfactants) are effective in increasing water infiltration, but they are costly and their use is limited largely to turf grass (Wallis & Horne, 1992), although increases in barley and lupin yields have been achieved in broadacre cropping systems when wetting

agents have been applied as a band in furrows above the seed (Blackwell pers. comm.). Roper (2004) proposed a biological approach to managing water repellency and isolated a range of bacteria including actinomycetes that are able to utilise waxes as sole carbon sources. Recently, Roper (2005) reported that the addition of lime to sands in field experiments reduced water repellency and increased the numbers of wax-degrading bacteria in the soil. In an alternative strategy, inoculation of water repellent soils with selected wax-degrading bacteria under controlled laboratory conditions resulted in significant improvements in soil wettability (Roper, 2004). This concurs with a report by Hallett et al. (2006) who found that a basidiomycetes fungus reduced water repellency when applied to soil. Further testing of some of our bacterial isolates inoculated into water repellent sandy soils in south-western Australia is reported here. In these field experiments, water repellency was monitored in soils inoculated with a range of wax-degrading bacteria both individually and in mixtures. Two levels of lime were superimposed to determine “inoculant by lime” interactions. Material and methods Water repellency was measured using the MED test (KING,

Remediation of water repellent soils 1981). Details of the procedure and rationale are described in ROPER (2005). Soils range from wettable (MED = 0) to severely water repellent (MED ≥ 4). A soil that is moist on collection may be potentially water repellent if after drying it resists water infiltration and has a positive MED. Experiment 1. Inoculation in the field – small scale A site at Anketell ∼20 km south of Perth (31◦ 57 S, 115◦ 51 E) in Western Australia was chosen for a small-scale trial to test the effect of inoculation of wax-degrading bacteria on the water repellency of sandy soils. At this site the soil was a water repellent (MED = 2.8), deep white sand coloured grey at the surface due to organic matter (Uc1.2; NORTHCOTE et al., 1975) with a pH in CaCl2 (0.01M; 1:2) of 4.8. In order to separate each treatment, metal boxes (0.5 m × 0.5 m and 0.12 m deep) were inserted into the soil so that ∼20 mm remained above ground. Soil cores (25 mm diameter × 50 mm depth) were collected from within the boxes to determine the baseline MED prior to inoculation on 2 June 1993. Seven separate cultures, described by ROPER (2004), were used in this experiment: 1 culture of Roseomonas sp. (5b), 1 culture of Streptomyces sp. (9a), 1 culture of Mycobacterium sp. (77ww1), 1 culture of Nocardia sp. (36a), 3 cultures of Rhodococcus spp. (66b, 73a, 83a). Each culture was grown in triplicate in 150 mL of GMS medium containing glucose (20 g/L), yeast extract (3 g/L) and mineral salts [(g/L): NaNO3 , 2; K2 HPO4 , 1; KH2 PO4 , 0.5; KCl, 0.1; MgSO4 .7H2 O, 0.5; CaCl2 , 0.01; FeSO4 .7H2 O, 0.01; Na EDTA, 0.0015 (AKIT et al., 1981)] for up to 5 days at 130 rpm and 30 ◦C on a New Brunswick incubator shaker (Innova 4300) until a heavy growth was observed. Afterwards, the cultures were concentrated by centrifugation at 6000 g for 20 minutes. The supernatant was removed and the pellet of culture was suspended in sterile distilled water (150 mL per culture) which was applied evenly to the soil in a box. There were 3 replicate boxes for each culture. To another set of 3 boxes 150 mL of distilled water only was applied (non-inoculated control). At fortnightly to monthly intervals following inoculation, a soil core (25 mm diameter × 50 mm depth) was collected from each box and prepared for measurement of MED. There were 12 sampling dates from just prior to setup on 2 June 1993 to 15 June 1994. Three months after inoculation, an additional soil core was collected to assess the recovery of the inoculants by spreading a small amount of soil (∼0.1 g) on the surface of agar plates containing the same constituents as the GMS medium used to grow the organisms. Plates were incubated at 30 ◦C and growth, based on colony characteristics, after 10 days was observed. During the course of the experiment, spray irrigation was used to provide an even supply of water throughout the year. The site was located in a Mediterranean climate with a winter rainfall pattern. In the dry summer months an average of 14 mm/day was applied in a 1-h timeframe. During the winter months the amount of spray irrigation was reduced to supplement rainfall. Soil within the boxes was maintained free of weeds during the experiment. Experiment 2. Inoculation in the field – large scale A site near Woogenellup (34◦ 31 S, 117◦ 50 E) in southwestern Australia was chosen for a large scale evaluation of inoculation of wax-degrading bacteria in the field. The soil was a shallow bleached sand underlain by ironstone gravel

S359 within 10 cm of the soil surface (Uc2.12; NORTHCOTE et al., 1975). The soil had a pH in CaCl2 (0.01M; 1:2) of 5.2 and was highly repellent (MED 3.7–4.0, with an average of 3.9). Prior to the experiment, the site was sown to Serradella (Ornithopus sativus cv. Cadiz), sown May 1999. The plots were 20 m × 13.3 m and each treatment was separated from each other by a 2 m buffer. Prior to inoculation with wax-degrading bacteria in September 1999, nutrients were applied to the entire site. Mineral fertilizer (DAPSZC; Summit Fertilizers) were applied at a rate (kg/ha) N (13.8), P (14.9), S (6.8), Cu (0.04), Zn (0.12), Mn (0.016) and Mo (6.4 ppm) and Premium Compost (Custom Composts, Nambeelup, Western Australia) was applied at 2 t/ha. In order to raise the Ca2+ content, agricultural lime (Bornholm AGLime, Western Australia) (neutralising value 70%) was applied at a rate of 1 t/ha to half the plots. The cultures were grown for 5–7 days in 2 litres of GMS medium described above in Experiment 1. A total of six cultures of actinomycetes, five cultures of Rhodococcus spp. (66b, 73a, 73ww, 83ww1, 85b) and one culture of Mycobacterium sp. (74b) were inoculated individually or in a mixture to the soil. Each inoculant treatment was replicated 12 times, 6 with lime applied to the soil and 6 without lime. 60 mL of concentrated culture suspension was used for each plot (equivalent to 2.25 L/ha of concentrated microbial suspension) and was diluted (∼1/40) with water prior to spraying onto the soil. The mixture of inoculants was prepared proportionately to give the same overall concentration of inoculant as the individual treatments. At the time of spraying, microbial samples from the spray were collected for plate counting the inoculants. Serial (decimal) dilutions to 10−8 were done in sterile distilled water and 0.3 mL of each dilution was spread on GMS agar plates, incubated at 30 ◦C and read at 10–14 days. From the time of inoculation, soil cores (25 mm diameter × 50 mm depth) were collected from each plot and water repellency (MED) was determined for each core as described above. There were 9 sampling dates from the day of inoculation on 15 September 1999 to 2 August 2000. An additional core was collected from each plot for determining the survival of inoculants. These samples were air dried for a minimum of 2 days and heated at 100 ◦C for 1 h to eliminate non-spore forming bacteria and select for actinomycetes. Further selection for actinomycetes was done using Bacillus phage prepared from soil collected from the field site, using the method described by KURTBÖKE et al. (1992). Heat-treated soil (1 g) was added to 99 mL of sterile distilled water containing 3 mL of Bacillus phage and mixed on a rotary shaker (Innova 4300) at 130 rpm and 28 ◦C for 1 h to facilitate infection and lysis of Bacillus spp. by the phage. Serial dilutions (decimal) to 10−4 were done in sterile distilled water and 0.3 mL of each dilution was spread on the surface of GMS agar plates containing the fungicide BenlateR (50 mg/L; Du Pont) to control fungal growth and on the same agar medium without glucose or Benlate (YMS). The plates were incubated at 28 ◦C for up to 28 days after which growth of inoculants was observed. The results were analysed using the statistical package Genstat (version 8.2, Lawes Agricultural Trust, Rothamsted Experiment Station, UK). A repeated measures ANOVA was used for each experiment with fixed-effect factors of “inoculant” (Experiment 1) and “inoculant” and “lime” (Experiment 2).

S360

M. M. Roper Table 1. Detection of inoculants in soil samples collected at various intervals after application to limed (L+) or unlimed (L–) soil in Experiment 2. Detection of the inoculant on either medium was recorded (+). Undetected were recorded (–).

Average water repellency (MED)

3.2

3.0

Inoculant 2.8

l.s.d. (P = 0.5)

73ww/L+ 73ww/L– 74b/L+ 74b/L– 66b/L+ 66b/L– 73a/L+ 73a/L– 83ww1/L+ 83ww1/L– 85b/L+ 85b/L– Mixture/L+ Mixture/L– Nil/L+ Nil/L–

2.6

2.4

Control 77ww1

9a

36a

73a

83a

5b

66b

Treatment

Fig. 1. Experiment 1 – water repellency (MED) averaged over the life of the experiment, of field soils at Anketell inoculated individually with cultures of wax-degrading bacteria. l.s.d. (P0.05 ) = 0.27. Bars below the l.s.d. are significantly different (P < 0.05) from the non-inoculated control.

Detection +/– 12-Oct-99 10-Nov-99 15-Dec-99 22-Mar-00 + + + + + + + + – – – – – – – – + + + – + + + – + + + – + + + – + + + + + + – – + + + – + + + – + + – – + + – – – – – – – – – –

Results

Experiment 2. Inoculation in the field – large scale Plate counting of the microbial suspension prior to spray inoculation, indicated that the numbers/ha equivalent of bacteria applied to each plot in the inoculated treatments ranged from 109 (73ww, 83ww1), 1010 (66b), 1011 (74b), 1012 (73a, 85b). In soil samples collected at various intervals after inoculation in September 1999, inoculants were detected in October and November 1999 (Tab. 1), but by March 2000 only 2 (Rhodococcus sp. 73ww and 83ww1) were detected. The impact of inoculants and lime application on water repellency averaged over the life of the experiment is shown in Fig. 2. A repeated measures ANOVA of data collected on 9 sampling dates between 15 September 1999 and 2 August 2000 indicated significant (P < 0.001) effects of “inoculant”, “lime application” and “inoculant by lime” interaction. Without the application of lime, only one inoculant (Mycobacterium sp. 74b) significantly reduced water repellency compared with the non-inoculated, non-limed control.

MED (+lime) MED (-lime)

l.s.d.(P = 0.05) 4.2

4.0

3.8

ur e ix t M

85 b

83 w w 1

73 a

66 b

74 b

73 w

w

3.6

C on tro l

Experiment 1. Inoculation in the field – small scale In this experiment, a repeated measures ANOVA of measurements made over 12 sampling dates between 2 June 1993 and 15 June 1994 indicated a significant inoculant effect (P < 0.001). Water repellency, averaged over the life of the experiment to take account of seasonal variation, is presented in Fig. 1. Of the seven inoculants only two resulted in significant reductions in water repellency (Rhodoccocus sp. 66b; Roseomonas sp. 5b). When soils were plated 3 months after inoculation of the soil, 6 of the 7 inoculants were recovered based on colony appearance but Nocardia sp. (36a) was not present.

Average water repellency (MED)

4.4

Inoculant

Fig. 2. Experiment 2 – interaction between inoculation and lime and their effect on water repellency (MED) averaged over the life of the experiment, of field soils at Woogenellup inoculated individually or in a mixture with cultures of Rhodococcus spp. (66b, 73a, 73ww, 83ww1, 85b) and one culture of Mycobacterium sp. (74b). l.s.d. (P0.05 ) for “inoculant by lime” interaction = 0.05. For “inoculant only” and “lime only” l.s.d. (P0.05 ) was 0.04 and 0.02 respectively (not shown).

However, there was a strong “inoculant by lime” interaction and, compared with their non-limed counterparts, lime application resulted in significant reductions in water repellency in treatments inoculated with Rhodococcus spp. (73ww, 66b, 73a, 83ww1, 85b) and the mixture. Only a small reduction in repellency resulting from lime application was seen with 74b compared with the non-limed inoculated treatment. Lime application alone significantly reduced water repellency in the non-inoculated control.

Remediation of water repellent soils Discussion Laboratory experiments under controlled moisture and temperature, reported by Roper (2004), indicated there was potential for remediation of water repellent soils by inoculation with wax-degrading bacteria. She measured a decrease in water repellency from a MED of 2.7 down to 1 with one isolate (73ww; Rhodococcus sp.) and a lesser improvement with another Rhodococcus sp. (66b) and a Mycobacterium sp. (74b). This led to field experiments, reported here, to further evaluate the impact of wax-degrading inoculants on water repellency. In Experiment 1 at Anketell, only 2 inoculants (Rhodococcus sp. 66b; Roseomonas sp. 5b) resulted in significant improvements in wettability compared with the non-inoculated control (Fig. 1). Each individual plot was small (contained in boxes 0.5 m × 0.5 m) and variability in the results was high (l.s.d. P0.05 = 0.27). In the much larger Experiment 2 at Woogenellup, water repellency was very even across the whole site and inoculants were applied at heavy rates (equivalent to 109 –1012 /ha). Compost and fertiliser were applied to support the inoculants in the nutrient-poor sands. Because actinomycetes were identified as the predominant group of wax-degrading bacteria in water repellent soils (Roper, 2004), lime was added to half the plots to raise pH and Ca2+ content to favour actinomycetes (Alexander, 1977). This led to a significant “inoculant by lime” interaction (P < 0.001) resulting in significant reductions in water repellency with all limed treatments containing 5 individual inoculants of Rhodococcus spp. and their mixture including Mycobacterium sp. (Fig. 2). Only 1 inoculant (Mycobacterium sp. 74b) significantly improved soil wettability without lime. Lime was applied at a very low rate (1 t/ha, 70% neutralising value), but considered alone, there was a significant difference between limed and non-limed soils. This concurs with results reported in Roper (2005) in which it was shown that, compared to untreated soils, the application of lime resulted in 1) a more rapid and sustained decline in water repellency following the commencement of the winter rains after the dry summer season, and 2) an increase in the size of populations of naturally-occurring wax-degrading bacteria. In the field experiments reported here there was common agreement with the laboratory experiments reported by Roper (2004) in which actinomycetes belonging to Rhodococcus spp. and Mycobacterium sp. were successful in reducing water repellency in soil. These organisms are known for their ability to metabolise a wide range of organic compounds as sole carbon sources for energy and growth (Williams et al., 1989; Walter et al., 1991). Furthermore, measurements of surface tension in the growth medium of these bacteria indicated that all of them produced biosurfactants (Roper, 2004). Biosurfactants emulsify hydrocarbons from soil surfaces and improve the efficiency of

S361 microbial degradation (Van Dyke et al., 1993). Actinomycetes are particularly at an advantage in soils because they form spores or resting stages that allow them to survive dry heat up to 110 ◦C (Nonomura & Ohara, 1969; Rowbotham & Cross, 1977). Despite this, the benefits in the field were much less than observed in the controlled laboratory experiments. Under field conditions inoculants introduced to bulk soil are subject to wetting and drying, large fluctuations in temperature and to competition from existing microbial populations. In Experiment 2 at Woogenellup, recovery of inoculants on agar media, based on colony appearance, was monitored over the first few months following spray-inoculation. Inoculants (particularly Rhodococcus spp.) were detected throughout the spring and early summer (mid-October to mid-December 1999). After the hot dry summer, typical of the Mediterranean climate of the region, only 2 inoculants (Rhodococcus spp; 73ww and 83ww1) were observed by late March 2000 (Tab. 1). The results indicate that, although inoculation of field soils with wax-degrading bacteria significantly reduced water repellency, the benefit was relatively small and unlikely to be economic. Better results may have been achieved at the Woogenellup site had the soils been inoculated earlier in the year at the time of the first rains after the dry summer season. Warmer soil temperatures at that time followed by winter rains may have resulted in more rapid and sustained microbial activity by the inoculants. Repeated inoculation on an annual basis may also provide further benefits. To conclude, this paper is the last of a series of articles describing the potential for soil microorganisms to reduce water repellency. Roper (2004) isolated, from a range of sources, bacteria that are able to utilise waxes as sole sources of carbon. Some of these isolates, belonging to the actinomycetes (2 isolates of Rhodococcus spp. and 1 of Mycobacterium sp.), significantly reduced soil water repellency in controlled laboratory experiments. Roper & Gupta (2005) developed a method to enumerate these bacteria in soil. Two approaches to utilising these bacteria to remediate water repellent soils were evaluated: 1. Inoculation of water repellent soils with the most effective wax-degrading bacteria; 2. Use of managements to promote populations of naturally-occurring, wax-degrading bacteria. Inoculation of water repellent soils, described in this paper, produced significant reductions in water repellency at 2 different sites. Rhodococcus spp. were the most successful inoculants overall. However, although statistically significant, improvements in wettability were relatively small, and the costs of production and application of the inoculant would likely outweigh the benefits. The addition of small amounts of lime (1 t/ha, 70% neutralising value) interacted favourably with inoculation and significantly increased soil wettability. More detailed studies on the impact of manage-

S362 ment practices on water repellency (Roper, 2005) indicated that the application of lime (3–5 t/ha) to water repellent soils increased the size of naturally-occurring populations of wax-degrading bacteria by up to an order of magnitude and, compared with non-limed controls, resulted in significant and substantial reductions in water repellency in the field for at least 4 years. Application of lime is simple and economic, and has other benefits such as raising soil pH to levels that are more suitable for microbial function in the soil and for plant growth. In this same study, it was shown that irrigation of water repellent soils maintained soil moistures at levels favourable for microbial activity and resulted in sustained reductions of repellency over time. These studies show that there is considerable potential to increase soil wettability through increased microbial activity by wax-degrading bacteria. Managements that increase wax-degrading activity by populations of naturally-occurring bacteria are likely to be more practical and economic than inoculation with selected wax-degrading bacteria. Bioremediation of water repellent soils provides an alternative approach to other more expensive strategies such as wetting agents or claying which may also be excluded if clays are unavailable on site. Acknowledgements This work was supported by Grains Research and Development Corporation. The author is grateful to Anne MCMURDO and to Cindy MYERS for technical assistance. The author thanks Grant and Helen COOPER, “Grassmere”, Woogenellup, and Doug and Debbie LIEVENSE, Anketell for the use of their land for field experiments. References AKIT, J., COOPER, D. G., MANNINEN, K.I. & ZAJIC, J.E. 1981. Investigation of potential biosurfactant production among phytopathogenic Corynebacteria and related soil microbes. Curr. Microbiol. 6: 145–150. ALEXANDER, M. 1977. Introduction to soil microbiology, 2nd ed. John Wiley and Sons Inc., New York, 467 pp. BLACKWELL, P. 1993. Improving sustainable production from water repellent sands. West. Aust. J. Agric. 34: 160–167. BOND, R.D. 1964. The influence of the microflora on the physical properties of soils II. Field studies on water repellent soils. Aust. J. Soil Res. 2: 123–131. FRANCO, C.M.M., TATE, M.E. & OADES, J.M. 1995. Studies on non-wetting sands. I. The role of intrinsic particulate organic matter in the development of water-repellency in non-wetting sands. Aust. J. Soil Res. 33: 253–263. FRANCO, C.M.M., CLARKE, P.J., TATE, M.E. & OADES, J.M. 2000. Hydrophobic properties and chemical characterisation of natural water repellent materials in Australian sands. J. Hydrol. 231–232: 47–58. HALLETT, P.D., WHITE, N.A. & RITZ, K. 2006. Impact of basidiomycetes fungi on the wettability of soil contaminated with a hydrophobic polycyclic aromatic hydrocarbon. Biologia, Bratislava 61(Supl. 19): S334–S337. KING, P.M. 1981. Comparison of methods for measuring severity of water repellence of sandy soils and assessment of some factors that affect its measurement. Aust. J. Soil Res. 19: 275–285.

M. M. Roper KURTBÖKE, D.I., MURPHY, N.E. & SIVASITHAMPARAM, K. 1992. Use of bacteriophage for the selective isolation of thermophilic actinomycetes from composted eucalyptus bark. Can. J. Microbiol. 39: 46–51. LICHNER, L., DLAPA, P., DOERR, S.H. & MATAIX-SOLERA, J. 2006. Evaluation of different clay minerals as additives for soil water repellency alleviation. Appl. Clay Sci. 31: 238–248. MA’SHUM, M., TATE, M.E., JONES, G.P. & OADES, J.M. 1988. Extraction and characterization of water-repellent materials from Australian soils. J. Soil Sci. 39: 99–110. MA’SHUM, M., OADES, J.M. & TATE, M.E. 1989. The use of dispersible clays to reduce water-repellency of sandy soils. Aust. J. Soil Res. 27: 797–806. MCKISSOCK, I., WALKER, E.L., GILKES, R.J. & CARTER, D.J. 2000. The influence of clay type on reduction of water repellency by applied clays: a review of some West Australian work. J. Hydrol. 231–232: 323–332. MCKISSOCK, I., GILKES, R.J. & WALKER, E.L. 2002. The reduction of water repellency by added clay is influenced by clay and soil properties. Appl. Clay Sci. 20: 225–241. MORLEY, C.P., MAINWARING, K.A., DOERR, S.H., DOUGLAS, P., LLEWELLYN, C.T. & DEKKER, L.W. 2005. Organic compounds at different depths in a sandy soil and their role in water repellency. Aust. J. Soil Res. 43: 239–249. NONOMURA, H. & OHARA, Y. 1969. Distribution of actinomycetes in soil. VI. A culture method effective for both preferential isolation and enumeration of Microbispora and Streptosporangium strains in soil (part 1). J. Fermentation Technol. 47: 463–469. NORTHCOTE, K.H., HUBBLE, G.D., ISBELL, R.F., THOMPSON, C.H. & BETTENAY, E. 1975. A description of Australian soils. CSIRO, Australia, 170 pp. RITSEMA, C.J., DEKKER, L.W. & HEIJS, A.W.J. 1997. Threedimensional fingered flow patterns in a water repellent sandy field soil. Soil Sci. 162: 79–90. ROBERTS, F.J. & CARBON, B.A. 1972. Water repellence in sandy soils of south-western Australia. II. Some chemical characteristics of the hydrophobic skins. Aust. J. Soil Res. 10: 35–42. ROPER, M.M. 2004. The isolation and characterisation of bacteria with the potential to degrade waxes that cause water repellency in sandy soils. Aust. J. Soil Res. 42: 427–434. ROPER, M.M. 2005. Managing soils to enhance the potential for bioremediation of water repellency. Aust. J. Soil Res. 43: 803– 810. ROPER, M.M. & GUPTA, V.V.S.R. 2005. Enumeration of waxdegrading microorganisms in water repellent soils using a miniaturised Most-Probable-Number method. Aust. J. Soil Res. 43: 171–177. ROWBOTHAM, T.J. & CROSS, T. 1977. Ecology of Rhodococcus coprophilus and associated actinomycetes in fresh water and agricultural habitats. J. Gen. Microbiol. 100: 231–240. TATE, M.E., OADES, J.M. & MA’SHUM, M. 1989. Non-wetting soils, natural and induced: overview and future developments. In: The theory and practice of soil management for sustainable agriculture. A workshop of the Wheat Research Council, Aust. Gov. Publishing Service, Canberra, A. C. T., Australia, pp. 70–77. VAN DYKE, M.I., GULLEY, S.L., LEE, H. & TREVORS, J.T. 1993. Evaluation of microbial surfactants for recovery of hydrophobic pollutants from soil. J. Industrial Microbiol. 11: 163–170. WALLIS, M.G. & HORNE, D.J. 1992. Soil water repellency. Adv. Soil Sci. 20: 91–146. WALTER, U., BEYER, M., KLEIN, J. & REHM, H-J. 1991. Degradation of pyrene by Rhodococcus sp.UW1. Appl. Microbiol. & Biotechnol. 34: 671–676. WARD, P.R. & OADES, J.M. 1993. Effect of clay mineralogy and exchangeable cations on water-repellency in clay-amended sandy soils. Aust. J. Soil Res. 31: 351–364. WILLIAMS, S.T., SHARPE, M.E. & HOLT, J.G. (eds) 1989. Bergey’s manual of Systematic Bacteriology, Vol. 4. Williams and Wilkins, Baltimore, pp. 2299–2648.