Effects of cations, temperature, and creosote on degradation of indole by Desulfobacterium indolicum (DSM 3383)

INTRODUCTION

The major contributor of heterocyclic compounds to the environment is creosote contamination at abandoned gasworks [1]. Creosote is a complex mixture consisting of approximately 3% heterocyclic compounds, 12% phenolic compounds, and 85% polycyclic aromatic hydrocarbons (PAHs) [2]. Many of the nitrogen-heterocyclic compounds are toxic [3-5], terato-genic [4], mutagenic [6, 7], and/or carcinogenic [8].

Desulfobacterium indolicum is a sulfate-reducing bacterium able to grow on the nitrogen-heterocyclic compounds indole and quinoline [9], two of the most abundant heterocyclic compounds in creosote [2]. Desulfobacterium indolicum was isolated from marine mud with indole as the sole electron donor and carbon source. The isolation was performed at 28°C and pH 7. The cultivation medium contained 360 mM NaCl and 23 mM MgCl² as well as 1.0 mM CaCl² and 6.7 mM KCl. Bak and Widdel [9] found that any changes in the concentrations of NaCl, MgCl2, or CaCl2 retarded the growth of D. indolicum. The cation concentrations in ground water are typically 0.1 to 2 mM of Na⁺, Mg²⁺, and Ca²⁺, and 0.01 to 0.1 mM of K⁺ [10].

In addition to indole and quinoline, D. indolicum can use the aromatic compounds anthranilic acid and 2-aminobenzoate as the sole electron donor and carbon source. However, growth with substrates other than indole is rather slow [9]. Shranker and Bollag [11] found that D. indolicum hydroxylated 95% of 0.13 mM of indole within 10 d, producing oxindole. The oxindole was subsequently transformed within 35 d.

The initial step of indole and quinoline degradation is hydroxylation [12-18]. The initial degradation step is less well known for other heterocyclic compounds. Some aromatic compounds are metabolized via a few central intermediates during anaerobic degradation, which maximizes the substrate spectrum of an organism and minimizes the number of enzymes required [19]. However, the substrate spectrum and the enzyme specificity during anaerobic degradation of heterocyclic compounds are unknown.

Examination of the characteristics of anaerobic organisms growing on heterocyclic compounds is of interest, as these compounds can be used for bioremediation. As a marine organism, D. indolicum has a need for cations. Therefore, definition of the need for cations, along with growth temperatures and utilization of other creosote compounds, is also of interest.

MATERIALS AND METHODS

RESULTS

Effect of cations

Only Na⁺ and Mg²⁺ had a significant effect (p < 0.05) on indole hydroxylation by D. indolicum, as shown by a statistical two-factorial analysis of the results done by the computer program Design-Ease [20]. Increased concentrations of Mg²⁺ partly compensated for a reduction of the Na⁺ concentration. Reduction of the concentrations of K⁺ and Ca²⁺ had no effect on the indole hydroxylation (Table 1). The experiment using media with normal concentrations of K⁺ and Ca²⁺, and different combinations of Na⁺ and Mg²⁺ concentrations, showed that indole hydroxylation had a linear response to cation concentrations (p < 0.01). The linear relationship was described by the following function (R² = 0.63):

Correspondingly, a conductivity of 20 to 40 mmho/cm generally resulted in a high indole hydroxylation rate (Table 2). No relationship was found between the conductivity and the hydrolylation rate (Tables 1 and 2). For example, indole degradation did not occur in a medium with a conductivity of 23 mmho/cm when no Mg²⁺ was present even though the medium contained 180 mM Na⁺. A medium containing 52 mM Na⁺ and 10 mM Mg²⁺ supported growth and degradation. However, the hydroxylation rates were reduced to about one third of normal rates. This medium composition was chosen for experiments with D. indolicum in cation-low medium.

Table Table 2.. Observed and predicted indole hydroxylation rates in media with 1.0 mM Ca²⁺ and 6.7 mM K⁺ and different combinations of Na⁺ and Mg²⁺

Sample No.	Na+ (mM)	Mg2+ (mM)	Conductivity (mmho/cm)	Observed degradation ratea (μmol/L/d)	Predicted degradation rateb (μmol/L/d)	Biomass (mg protein/L)c
1	307	58	37.5	85 ± 8.0	103	1.8
2	180	34	25.9	82 ± 8.0	63	2.0
3	180	68	29.8	79 ± 5.2	87	2.0
4	180	34	26.2	88 ± 6.3	63	2.0
5	180	34	26.3	84 ± 1.4	63	1.7
6	360	34	39.2	80 ± 3.1	97	2.0
7	52	58	19.4	55 ± 5.1	56	2.1
8	180	34	26.0	77 ± 1.4	63	1.8
9	0	34	12.2	12 ± 1.0	30	1.3
10	307	10	32.8	86 ± 9.1	70	2.0
11	180	0	22.6	6 ± 2.2	39	0.7
12d	52	10	13.8	23 ± 1.1	23	0.8
Normal medium	360	23	41.3	69 ± 8.6	88	1.5

• ^a Observed rate of hydroxylation estimated by linear regression ± standard error. Initial concentration of indole was 100 μM.
• ^b Predicted degradation rate of indole from the equation: degradation = 2.5 + 0.19·(mM Na⁺) + 0.76·(mM Mg²⁺).
• ^c Biomass quantified as protein after 21 d.
• ^d Medium composition chosen as cation-low medium.

Effect of creosote mixture

Indole was hydroxylated at the same rate in all cultures with 0 to 40 mg/L of the creosote mixture added to medium with normal concentrations of cations. Indole hydroxylation was 30% inhibited in cultures with 60 mg/L creosote, and 96% inhibited in cultures with 100 mg/L (Table 3).

Indole was hydroxylated at the same rate in cultures with 0 to 20 mg/L of creosote in cation-low medium, where the salt concentrations were reduced to 52 mM Na⁺ and 10 mM Mg²⁺. However, 86% inhibition was observed in cultures with 60 mg/L of creosote added, corresponding to only 7% of the hydroxylation rate observed in the medium with normal salt content and with the same concentration of creosote added. Degradation stopped before indole was completely used in all cultures in the cation-low medium, including the medium with no creosote present. The indole hydroxylation was generally stimulated by low concentrations of creosote. Measurements showed that indole and quinoline were the only compounds from the creosote mixture that were degraded.

Table Table 3.. Hydroxylation rates and relative hydroxylation rates of indole in the normal medium and in the cation-low medium

	Normal mediuma		Cation-low mediumb
Concentration of creosote mixture (mg/L)	Initial hydroxylation rate (μmol/L/d)c	Relative hydroxylation rate (%)d	Initial hydroxylation rate (μmol/L/d)c	Relative hydroxylation rate (%)d
Sterile control	0	0	0	0
No creosote added	39 ± 2.9	100	13.3 ± 0.8	100
5 mg/L	44 ± 3.7	112	ND	ND
10 mg/L	44 ± 2.3	112	14.2 ± 0.9	107
20 mg/L	45 ± 3.2	115	12.8 ± 0.9	96
40 mg/L	40 ± 4.0	103	7.1 ± 0.9	53
60 mg/L	27 ± 4.4	69	1.8 ± 0.9	14
100 mg/L	1.7 ± 1.5	4	ND	ND

• ^aMedium contains 360 mM Na⁺ and 23 mM Mg²⁺.
• ^bMedium salt reduced to 52 mM Na⁺ and 10 mM Mg²⁺.
• ^c Hydroxylation rate of indole estimated by linear regression of the first 4 d ± standard error.
• ^d Hydroxylation rate relative to the hydroxylation rate in a medium with no creosote added. ND: Not determined.

DISCUSSION

The cation content of the medium could be reduced considerably and still support growth of D. indolicum. The significant cations were Na⁺ and Mg²⁺, which could be reduced by about 15 and 60%, respectively, without reduction of the indole hydroxylation rate. The effect of reduced concentrations of cations did not reflect the lowering of the conductivity in the medium as there was no correlation between the conductivity and the hydroxylation rate. A reduction by 85 and 57% of Na⁺ and Mg²⁺, respectively, reduced the indole hydroxylation rate to one-third of normal rates. This result contradicts those of Bak and Widdel [9], who observed that any changes in the NaCl, MgCl₂, or CaCl₂ concentrations retarded the growth of D. indolicum. Lowering the temperature partly relieved the stress of lowering the cation concentration.

Madsen et al. [24] investigated the environmental factors affecting indole metabolism in denitrifying and methanogenic soil, sediment, and sewage sludge, and found that the indole degradation rate increased with temperatures from 15 to 35°C. Furthermore, they found nearly a threefold decrease in the maximum concentration of the intermediary product, oxindole.

Desulfobacterium indolicum was stressed by reduced salt concentrations, as seen from the result with creosote: creosote was approximately five times more toxic in the cation-low medium than in the medium with normal salt concentrations. The stimulation of indole degradation at low creosote concentrations might be due to a higher concentration of indole, 100 μM indole added, plus the indole from the creosote mixture. Indole and quinoline were the only compounds from the creosote mixture that were degraded, confirming results from Bak and Widdel [9] that D. indolicum only uses a restricted spectrum of aromatic carbon sources.

Toxicity studies of creosote compounds have shown that the heterocyclic compounds in creosote are the most toxic [25]. These studies found that addition of 38.9 mg of creosote mixture per liter of aerobic enrichment culture derived from groundwater inhibited toluene degradation by approximately 38% compared to cultures with only toluene added. The creosote mixture used by Dyreborg et al. [25] contained 55% heterocyclic compounds, which might explain the relatively high inhibition.

The experiments with addition of the creosote mixture to cultures of D. indolicum showed that D. indolicum did not transform pyrrole, carbazole, benzothiophene, dibenzothiophene, furan, benzofuran, or dibenzofuran, which are all structurally related to indole and quinoline. The inability of D. indolicum to transform these compounds indicates that they are either not hydroxylated or that they are hydroxylated by an enzyme system not active for indole and quinoline.

We have found no previous reports of the effect of cations on degradation of creosote. However, the degradation of crude oil by a mixed bacterial community isolated from a marine sediment was investigated by Mille et al. [26]. They found that the amount of oil degraded increased with increasing salt concentrations, to a maximum at 400 mM Na⁺. Biodegradation occurred in the 100 to 2,000 mM Na⁺ range. Each fraction of the crude oil was degraded after a 30-d incubation period at an NaCl concentration equivalent to seawater (approx. 400 mM).

Our results indicate that D. indolicum will have a high survival under conditions expected to be found at creosote contaminations. Furthermore, the bacterium will be active at quite low temperatures and has a high tolerance towards creosote. However, cation concentrations required for growth of D. indolicum are higher than those normally found in soils and groundwaters and could limit the use of this bacterium for bioremediation. Further, the substrate spectrum of D. indolicum was limited. However, the substrates used were removed at relatively high rates even though the initial hydroxylation is an energy-requiring step. Our general impression is that bioaugmentation of soils by addition of specific degrading bacteria will demand very high amounts of inoculum [27], making this addition less attractive. Therefore, a pump and treat method for the most water-soluble creosote compounds in a bioreactor with immobilized cells would be a more interesting approach.