H.M.STRATTON, R.J.SEVIOUR and J.A.SODDELL

Biotechnology Research Centre, La Trobe University College of Northern Victoria, P.O.Box 199,

Bendigo, Victoria, Australia.

Code Number: AC93006
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ABSTRACT. Cells of several isolates of Nocardia, Rhodococcus and Mycobacterium were hydrophobic as determined by the microbial adherence to hydrocarbons (MATH) assay. However their hydrophobicity varied with both culture age and the media in which they were grown. The possible role of cell surface hydrophobicity in the ability of some of these isolates to cause and stabilize foams in activated sludge plants is discussed.

Many microbial cells are hydrophobic, and cell surface hydrophobicity (CSH) is often an important determinant in an organisms ability to colonize host tissue (Mamo, Rozgonyl and Brown, 1987; Hazen, 1989; Drumm et al., 1989) or inanimate materials (Marshall, 1976; Fattom and Shilo, 1984), and selectively accumulate at air/liquid interfaces (Bezdek and Carlucci, 1972; Kjelleberg et al., 1976). This hydrophobicity appears to be imparted by different chemical components of the cell wall in different bacteria, and these include e lipoteichoic acids (Mioner et al., 1983) and proteins (Parker and Munn, 1984). The nocardioform bacteria have a unique cell wall chemistry containing mycolic acids (Blackall, 1987; Mori et al., 1987) whose presence might render their cells hydrophobic. However, little information is available on the CSH of nocardioforms other than Nocardia amarae, which was shown to be hydrophobic when grown on several different carbon sources (Blackall and Marshall, 1989).

It is known that foams forming on the surface of activated sludge plants often contain nocardioforms (Soddell and Seviour, 1990) and their hydrophobicity might be important in enabling them to attach to gas bubbles and float (Lemmer, 1986). Whether these foam isolates are hydrophobic and whether this hydrophobicity might change with changes in culture conditions is not known. In trying to explain their presence in foams, this might be an important consideration. Therefore this study was undertaken to see if several genera of nocardioforms bacteria were hydrophobic, and whether CSH changed in response to changes in the organisms environment.

MATERIALS and METHODS

Organisms. The nocardioform isolates used in the study including some which were obtained from foams from a number of activated sludge systems, are listed in Table 1. Those isolates prefixed J were isolated and identified by one of us (JS). All cultures were maintained on Bacto Standards Methods Agar (SMA) (Difco) at 20-22 C.

Effect of Culture Age on Cell Surface Hydrophobicity. Cells were grown in a yeast extract (YE) medium containing 3.0g/l Yeast Extract (Oxoid) and 5.0g/1 Bacteriological Peptone (Oxoid), pH 7.0, and the medium was dispensed as 150 ml aliquots in 500ml Erlenmeyer flasks.

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Organism     UCNV Culture     Source                
Hydrophobicity
Number
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Nocardia sp.    J 81     Foam isolate, Tamworth, NSW   ++
N.amarae        CON 9    Foam isolate, UQ*             ++
N.caviae        CON 14   AMMRL 19.11                   ++
N.asteroides    CON 4    UQ                            ++
N.pinensis      J 20     Foam isolate, Mildura, Vic    -
Nocardioform    J 27     Foam isolate, Delungra, NSW.  ++
Rhodococcus sp. J 32     Foam isolate, Jenolan Caves, NSW. ++ 
R.rhodochrous   CON 11R  Foam isolate, UQ*             ++
R.rhodochrous   CON 3    UQ                            -
R.erythropolis  CON 19   UQ                            ++
R.coprophilus   CON 18   UQ                            ++
M.smegmatis     CON 1    Unknown                       ++
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Inocula were prepared using 7-14d old cultures grown on SMA. Cells were suspended in 5ml sterile water to give a cell turbidity equivalent to McFarland tube 6 (Hendrickson, 1985) and 1ml aliquots of this suspension were used to inoculate the culture media, which were incubated in an orbital shaker (Paton Industries, South Australia) at 190rpm and 26 C. Population growth was monitored by changes in absorbance at 540nm, and cells were harvested during early, mid, late exponential growth and at stationary phase. Their CSH was then determined as described below. All experiments were repeated at least twice, and values are the means of 3 determinations for each experiment.

Effect of Medium Composition on CSH. In the experiments where the effects of varying Carbon:Nitrogen (C:N) ratios on CSH were assessed, the concentration of C source was varied, while that of the N source was kept constant. The isolates selected were N.amarae, R. rhodochrous (CON llR) and Rhodococcus sp. (J 32). The medium used contained 1.6g/l Bacto Yeast Nitrogen Base Without Amino Acids and Ammonium Sulphate (YNB) (Difco), 4.7g/l (NH4)2S04 and 0.006g/l Bacto Casamino Acids (Difco), and was neutralised with 13.0mM K2HPO4. The basal medium and buffer were sterilised separately at 121 C for 15min. Differing C:N ratios were achieved by adding 12.5g/l, 25g/l and 50g/l glucose (sterilised separately by membrane filtration) to give final C:N ratios of 5:1, 10:1 and 20:1, respectively. These media were dispensed, inoculated and cells were harvested as described above. Cell surface hydrophobicity was determined as described below.

Determination of Cell Surface Hydrophobicity. The method used for cell surface hydrophobicity determinations was the microbial adherence to hydrocarbons (MATH) assay, with n-hexadecane as the solvent (Rosenberg et al., 1980).

Depending on the culture turbidity, a 10, 15, 20 or 30ml sample was taken for the assay, and the percentage CSH was then determined using the method of Hogg and Manning (1987). Other methods for measuring CSH were considered less suitable for these filamentous bacteria. For example, methods such as Hydrophobic Interaction Chromatography (Smyth et al., 1978) are not appropriate because of possible physical retention of filamentous cells and the Salt Aggregation Test (Lindahl et al., 1981) is subjective and not quantitative.

Figure 1. Cell surface hydrophobicity of isolates at different stages of their growth cycle as measured by the MATH assay, with n-hexadecane as the solvent. Each experiment was repeated and the data points are the average of six readings taken over two experiments. The results for nocardioform J 27 are an average of three. The error bars represent the standard deviation. Other details are in the text.

RESULTS

Effect of Culture Age on CSH. The nocardioform isolates tested exhibited strong hydrophobic behaviour at some stage in their growth cycle on YE Medium (Fig. 1), but levels of CSH varied between strains, and often with culture age. Mean doubling times (td) of these isolates also differed, as did the duration of their lag phases (Table 2), but neither of these variables showed, any direct correlation with culture CSH. For example, the Rhodococcus sp., (td 16 h) was very hydrophobic while strain J 27 which grew extremely slowly (td 50 h) also had high but more variable CSH. Both the R.rhodochrous isolates tested were relatively rapid growers with low td values, but CON 11R showed high levels of CSH while CON 3 was only weakly hydrophobic. The Rhodococcus sp. (J 32), and N.asteroides, both had consistently high hydrophobicity at all stages of their growth cycle. R.rhodochrous (CON 11R) was hydrophobic during exponential growth, increasing in stationary phase, yet R.rhodochrous (CON 3) showed the opposite trend. Hydrophobicity of both Nocardia sp. and M.smegmatis increased during exponential growth and remained high, while the nocardioform (J 27) from foam was also hydrophobic but levels fluctuated throughout its growth cycle.

Effects of Changing C:N Ratios on CSH. The td of the three foam isolates selected for these experiments, N.amarae, R.rhodochrous (CON 11R) and Rhodococcus sp. (J 32), were all lower in the YNB media than in the YE medium (Table 3), but again there appeared to be no consistent relationship between CSH and high or low growth rates. For example, although N.amarae grew extremely slowly in the YNB media and relatively rapidly in the YE medium its CSH was comparably high in both. At the higher C:N ratios of 10:1 and 20:1, lower CSH values were found in all three isolates than at 5:1 and this was most marked with Rhodococcus sp. (J 32) where CSH was close to zero (Fig. 2). These data clearly show that in CSH may change markedly in response to different C:N ratios, but these changes did not consistently occur at any particular stage in the growth cycles of all isolates.

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                     Duration of   Mean doubling   Time to Reach 

Organism              lag Phase        Time        Stationary
Phase
                      (hrs)          (hrs)        (hrs)
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Rhodococcus sp.           24            16            64
R.rhodochrous (CON 11R)   32             9            64
R.rhodochrous (CON 3)     40            10            68
M.smegmatis               48            22           100
N.amarae                  48            16            90
N.asteroides              64            18           198
Nocardia sp. (J 81)       60            12           100
Nocardioform (J 27)      168            50           336
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Table 2. Kinetic parameters of a selection of nocardioform isolates grown in YE medium. Other details are given in text.

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Organism     Duration of Lag   Mean Doubling  Time to reach     

                 Phase            Time        Stationary phase
                 (hrs)            (hrs)           (hrs)
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C:N Ratios       5:1  10:1  20:1  5:1  10:1  20:1  5:1  10:1  20:1 
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N.amarae        84   84   120    59   53    70   228   222   200  
R rhodochrous   78   72    64    15   18    28   144   120   94  
(CON 11R) 

Rhodococcus     24   18    24    25   26    27    72    72   72 
sp. (J32)
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Table 3. Kinetic parameters Or a selection of nocardioform isolates grown in YNB medium at different C:N ratios. Other details are given in text.

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Figure 2. Cell surface hydrophobicity of selected isolates at different stages of their growth cycle grown with glucose as the sole carbon source in response to changing C:N ratios (square: 5:1; circle: 10:1; triangle: 20:1).

DISCUSSION

As expected, all the nocardioform isolates examined here were hydrophobic under some of the conditions examined, although the considerable variations seen in CSH at the different culture ages and C:N ratios were less expected.

Surface hydrophobicity in these organisms is assumed to be due to the presence of large amounts of mycolic acids in their cell walls (Mori et al., 1988) but it can be imparted by other surface components in nocardioforms like the polysaccharides in Rhodococcus sp. (Neu and Poralla 1988). Mycolic acid chemistry varies with genus, and carbon chain length is considered an important determinant of surface hydrophobicity (Mori et al., 1988), a longer carbon chain length providing a more hydrophobic surface. This may partly explain why Nocardia spp. (chain length 4460) were usually more hydrophobic than the Rhodococcus spp. (chain length 34-52) (Soddell and Seviour, 1990) examined under these culture conditions. If that were the case then the mycolic acid composition of these cells would vary with changes in the environment, as has been shown in other Rhodococcus spp. (Mori et al., 1988). It is not yet known if similar changes occur in these strains.

Surprisingly few studies have considered whether CSH changes with culture age or growth rate in bacteria, but culture age appears to be an important variable and in one such study, the hydrophobicity of a Chromobacterium sp. and Flexibacter sp. was shown to change with both culture age and different limiting carbon sources (McEldowney and Fletcher, 1986). Thus, these assays should be carried out on cells at the same stages of their growth cycles under strictly defined culture conditions, which is rarely satisfied in many published reports. It may also be wise to microscopically check the aqueous/hydrocarbon interface at the completion of the assay, since absorbance of the aqueous phase may not be solely due to the presence of cells. This was the case with Rhodococcus sp. (J 32) in the YNB medium, with cells clearly adhering to the hexadecane droplets, although the aqueous phase remained turbid, possibly due to the formation of an emulsifying agent. The Rhodococcus described by Neu and Poralla (1988) behaved similarly, due to the presence of a polysaccharide capsule, and the mucoid appearance of J 32 on solid media (unpublished) may also be associated with the production of a similar emulsifier.

The culture conditions used here do not closely simulate those found in activated sludge plants. However, the generally high surface hydrophobicity of the foam isolates is consistent with their possible involvement in foam formation. It might be useful to explore possible relationships between nutrient status of waste water treatment plants and biomass CSH, as our results suggest that these may change with variations in operating conditions, like carbon sources or varying nutrient limitations. The little published data on the role of hydrophobicity in foaming activated sludge plants has not considered nutrient status of the mixed liquor as a possible important variable. For example, although Khan and Forster (1988) showed that mixed liquor biomass was usually more hydrophobic in foaming plants than in non-foaming plants, and in most cases CSH of foam solids was greater than those from mixed liquor, nutrient status of the mixed liquor was not known and neither were the foam causing organisms identified.

What level of hydrophobicity is required by a population of cells to behave as hydrophobic particles in the formation of foam is not known, but other factors may also be important. Surfactants produced by nocardioforms and other organisms in activated sludge (Kahn and Forster, 1988) or entering treatment plants (Ho and Jenkins, 1991) may also contribute to the flotation process that ultimately expresses itself as foaming in aerated reactors. Unfortunately, what little information is available on the role of mixed liquor nutrient status on foam formation in activated sludge reactors is still inadequate to allow design of sensible biological foam control measures. This will only come when our understanding of its microbiology increases.

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