Degradation of Aromatic Compounds Coupled to ... - Microorganisms

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VICTORIA K. KNIGHT ... New Brunswick, New Jersey, USA ... Rutgers, The State University of New Jersey, 76 Lipman Drive, New Brunswick, NJ 08901-8525.
Geomicrobiology Journal, 19:77 – 86, 2002 C 2002 Taylor & Francis Copyright ° 0149-0451 /02 $12.00 + .00

Degradation of Aromatic Compounds Coupled to Selenate Reduction VICTORIA K. KNIGHT IVONNE NIJENHUIS Biotechnology Center for Agriculture and the Environment Rutgers, The State University of New Jersey New Brunswick, New Jersey, USA

LEE J. KERKHOF Institute of Marine and Coastal Sciences Rutgers, The State University of New Jersey New Brunswick, New Jersey, USA

¨ MAX M. HAGGBLOM Department of Biochemistry and Microbiology Biotechnology Center for Agriculture and the Environment Rutgers, The State University of New Jersey New Brunswick, New Jersey, USA The degradation of a range of aromatic compounds under selenate-reducing conditions was examined. Enrichment cultures were initiated with a 10% sediment slurry from the Arthur Kill, an intertidal strait in the New York/New Jersey harbor, and from the Kesterson Reservoir, California, with selenate as the sole electron acceptor. After repeated feeding, activity was maintained with either benzoate, 3-hydroxybenzoate, or 4-hydroxybenzoate as the sole carbon substrate. Degradation of the benzoic acid derivatives did not occur without the addition of selenate, or in sterile controls, indicating that substrate utilization was dependent on selenate reduction. Degradation of each of these aromatic compounds was coupled stoichiometrically to the reduction of selenate to selenite. Two selenate-reducing bacterial strains that utilized 4-hydroxybenzoate as a carbon source were isolated from the Arthur Kill and the Kesterson Reservoir, respectively. Based on analysis of their 16S rRNA gene sequences these isolates were 98.3% similar to one another, but distinct from other known Se(VI)-reducing bacteria. Phylogenetic analysis indicated that they were afŽ liated with the gamma subgroup of the Proteobacteria, with uncultured sulfur-oxidizing symbionts as their closest neighbors. Keywords

anaerobic degradation, selenate reduction, benzoate, hydroxybenzoate

Received 12 May 2001; accepted 9 October 2001. We thank R. Oremland, USGS (Menlo Park, CA), and Beau Ranheim and the crew of the Osprey (NYC-DEP) for providing the sediment samples from Kesterson Reservoir and Arthur Kill, respectively. We thank L. Y. Young for use of shared analytical instrumentation. This work was supported in part by the U.S. Environmental Protection Agency (Grant R822487) and the OfŽ ce of Naval Research (Grants N00014-94-1-043 4 and N-00014-97-1-076 1). Address correspondenc e to: Max M. Ha¨ ggblom, Department of Microbiology and Biochemistry, Rutgers, The State University of New Jersey, 76 Lipman Drive, New Brunswick, NJ 08901-8525 . E-mail: [email protected]

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Introduction Selenium contamination of agricultural wastewaters draining from seleniferous soils is a widespread problem in the Western United States (McNeal and Balistrieri 1989; Presser 1994). Irrigation water applied to agricultural soil dissolves selenium as selenate [Se(VI)] and selenite [Se(IV)] oxyanions. Due to their toxicity, these selenium oxyanions must be removed from water before release into the environment (Doran 1982; Presser 1994). Drainage waters are often treated by transferring them to ponds for storage and evaporation. In the San Joaquin Valley California, approximately 160,000 ha of farmland are affected by salinity, selenium, and high water tables (Squires et al. 1989). Total selenium concentrations as high as 3,000 ¹g/l often occur in these shallow saline groundwaters (McNeal and Balistrieri 1989; Squires et al. 1989). Transformation of selenium in the environment is primarily microbially mediated (for reviews see Doran 1982; Oremland et al. 1989; Macy 1994; Oremland 1994; Stolz and Oremland 1999). 2¡ Microbial reduction of toxic selenium oxyanions (SeO2¡ 4 and SeO3 ) to less toxic and insoluble elemental selenium is the predominant mechanism for selenate removal from aqueous systems. Under aerobic conditions, a diverse group of bacteria can reduce selenate (Maiers et al. 1988; Lortie et al. 1992, reviewed in Stolz and Oremland 1999); however, this is thought to be primarily a detoxiŽ cation strategy. Under anaerobic conditions several sulfatereducing bacteria (Tomei et al. 1995; Zehr and Oremland 1987), Wolinella succinogenes (Tomei et al. 1992), and Enterobacter cloacae (Losi and Frankenberger 1997) are capable of reducing micromolar amounts of selenate but do not couple this reduction to growth. In addition, phototrophic bacteria are also able to reduce selenate from redox poise created during anaerobic growth (Moore and Kaplan 1994; Yamada et al. 1997). In contrast, a number of bacteria are able to grow anaerobically by dissimilatory selenate reduction (Fujita et al. 1997; Knight and Blakemore 1998; Macy et al. 1989, 1993; Oremland et al. 1994; Switzer Blum et al. 1998). These organisms are able to reduce millimolar amounts of selenium oxyanions. Most selenate-reducing bacteria are metabolically diverse and are able to couple growth to the reduction of a wide range of alternate terminal electron acceptors including metals (Macy et al. 1993; Laverman et al. 1995; Knight and Blakemore 1998; Stolz and Oremland 1999). In addition, bacteria have been characterized that grow by dissimilatory selenite reduction (Macy et al. 1989; Switzer Blum et al. 1998). It is interesting to note that complete reduction of Se(VI) to Se0 often requires a mixed culture whereby one organism reduces Se(VI) to Se(IV) and a second organism reduces Se(IV) further to Se0 (Macy et al. 1989; Switzer Blum et al. 1998). All of the previously described dissimilatory Se(VI)-reducing bacteria were isolated on aliphatic organic acids (acetate, ethanol, lactate) as the carbon source and the biodegradation of aromatic compounds coupled to selenate reduction has not been examined in detail. However, Thauera selenatis is able to grow aerobically on benzoic acid (Macy et al. 1993), and several closely related members of the Thauera genus are able to grow on diverse aromatic compounds under denitrifying conditions (Anders et al. 1995; Song et al. 1998, 2001). This study was undertaken to determine the extent of degradation of aromatic compounds coupled to the reduction of selenate.

Materials and Methods Source of Inoculum and Preparation of Enrichment Cultures Sediment grab samples were collected from the Arthur Kill, an intertidal strait between Staten Island, New York, and New Jersey and from the Kesterson Reservoir, California. Glass jars were Ž lled to capacity with sediment, sealed, and stored at 4± C until used.

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Enrichment cultures with a 10% (v/v) sediment inoculum were prepared following strict anaerobic techniques in minimal salts medium with 10 mM Na2 SeO4 as the terminal electron acceptor, with N2 :CO2 (70:30) as the head space gas, and Na2 S (98 ¹M) as reducing agent (Monserrate and Ha¨ ggblom 1997; Knight et al. 1999). Sterile controls were autoclaved three times (30 min at 121 ± C) on consecutive days. Controls with no added substrate were prepared to determine background levels of Se(VI) reduction due to degradation of organic compounds in the sediment inoculum. Aromatic substrates (purity >97%, Aldrich Chemical Company, Milwaukee, Wisconsin) were added from sterile anaerobic stock solutions prepared in 0.1 N NaOH. Cultures were incubated in the dark at 28± C. Analytical Techniques Liquid samples were taken periodically for analysis by high performance liquid chromatography as described previously (Knight et al. 1999). A 4.6 £ 250 mm Ultrasphere C18 column (Beckman) was used with a mobile phase consisting of methanol:water:acetic acid (60:38:2, v:v:v) or (50:48:2, for hydroxybenzoates) at a  ow rate of 1 ml min¡1 . Selenium oxyanions were measured using ion chromatography (Dionex model DX-100; Dionex, Sunnyvale, California) with an AS9-SC (Dionex) column and an eluent of 1.8 mM Na2 CO3 : 1.7 mM NaHCO3 at a  ow rate of 2.0 ml min¡1 . Protein was measured using the Biorad assay (Biorad, Hercules, California) with bovine serum albumin as the standard. Samples were boiled in NaOH (0.1 N) for 10 min to solubilize protein prior to analysis. Isolation of Axenic Cultures Selenate-reducing strains were isolated from 4-hydroxybenzoat e degrading enrichment cultures using anaerobic soft agar shake tubes containing minimal salts medium supplemented with 10 mM Se(VI), 1 mM 4-hydroxybenzoate , and 4 g ¢ l¡1 noble agar (Difco, Detroit, Michigan). Isolated colonies were transferred multiple times in shake tubes and purity was veriŽ ed by observation of colony morphology and by microscopy. Isolates were transferred to liquid culture to verify loss of 4-hydroxybenzoat e and reduction of Se(VI). DNA Analysis DNA was extracted from active liquid cultures using a modiŽ cation of a phenol:chloroform method as previously described (Kerkhof and Ward 1993; Knight et al. 1999). Next, 16S rRNA was ampliŽ ed using standard eubacterial primers 27 F and 1525 R. PCR products were puriŽ ed using QIAquick PCR puriŽ cation kit (Qiagen, Santa Clara, California), sequenced, and phylogenetic trees were constructed from unambiguously aligned sequences as described previously (Knight et al. 1999).

Results and Discussion Utilization of Aromatic Compounds Under Se(VI)-Reducing Conditions Anaerobic enrichment cultures were established that degraded aromatic compounds under Se(VI)-reducing conditions from two ecologically different sites. The Arthur Kill is an intertidal strait in the New York Harbor with a sulfate concentration of approximately 20 mM. This site has previously been used to establish anaerobic enrichment cultures that degrade a wide range of aromatic compounds under different electron accepting conditions (Kazumi et al. 1995; Monserrate and Ha¨ ggblom 1997). In comparison, the Kesterson Reservoir site has high concentrations of selenium oxyanions as well as sulfate and nitrate (Presser 1994). Initial activity with enrichments from both sites were shown on a range of phenol and benzoic

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acid derivatives (data not shown). Benzoate and all three hydroxybenzoate isomers (100 ¹M) were utilized in the enrichments from Arthur Kill and Kesterson Reservoir. Utilization of benzoate, 3-hydroxybenzoate , and 4-hydroxybenzoat e was most rapid, with complete loss observed within 15 to 26 days, yet loss of 2-hydroxybenzoat e occurred within 68 to 75 days. Phenol and all of the mono-methylphenol isomers were utilized in the Arthur Kill enrichments within 15 to 58 days. None of the monobrominated phenols and benzoic acids, bromoxynil, 2,4,-dichlorophenox y acetic acid, and 4-nitrophenol was degraded under Se(VI)reducing conditions within 120 days. No loss of substrate occurred in the sterile controls. Benzoate, 3-hydroxybenzoate , and 4-hydroxybenzoat e were utilized upon refeeding of substrate, and stable Se(VI)-reducing enrichment cultures were maintained with inoculum from both sites by repeated subculturing into fresh medium and feeding of the respective substrates. The degradation of benzoate, reduction of Se(VI), and production of Se(IV) by enrichment cultures from Kesterson Reservoir is shown in Figure 1. No degradation of substrate or reduction of Se(VI) occurred in sterile controls. Similar results were obtained with 3-hydroxybenzoate , 4-hydroxybenzoate , and benzoate from both sites (data

FIGURE 1 Utilization of benzoate (A) and reduction of selenate (B) by enrichment cultures initiated with Kesterson Reservoir sediment. Symbols: (A) benzoate , benzoate sterile control r; (B) Se(VI) , Se(IV) ", Se(VI) sterile control H, Se(IV) sterile control r. Data points are the means of triplicate cultures § standard deviation.

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not shown). The difference between the amount of Se(VI) reduced and Se(IV) produced is presumably due to reduction of Se(IV) as evidenced by the formation of red amorphous Se0 in the cultures. Degradation of Benzoic Acid Derivatives Coupled to Se(VI)-Reduction To determine whether the degradation of benzoate, 3-hydroxybenzoate , and 4-hydroxybrea k benzoate was dependent upon the presence of Se(VI), enrichment cultures were transferred (1:5) into fresh medium with and without the addition of 5 mM Se(VI). Washing the enrichment cultures with sterile Se(VI)-free medium to remove Se(VI) irreversibly inhibited activity and therefore this step was eliminated from these experiments. Degradation of 3-hydroxybenzoat e was sustained only with the addition of Se(VI) as shown in Figure 2. A similar requirement for Se(VI) was demonstrated for anaerobic degradation of benzoate and 4-hydroxybenzoat e (data not shown).

FIGURE 2 Utilization of 3-hydroxybenzoat e (A) and reduction of selenate (B) by stable enrichment cultures from Arthur Kill sediment with and without the addition of Se(VI). Symbols: (A) 3-hydroxybenzoat e with added Se(VI) or without added Se(VI) ¤; (B) Se(VI) , Se(IV) " in selenate amended cultures, and Se(VI) ¥, Se(VI) ¤ in unamended controls. Data points are the means of triplicate cultures. No loss of substrate or reduction of selenate occurred in sterile controls.

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37 § 6 27 § 6 42 § 2

10 § 1 7§0 8§1

Carbon conversion to cellsb (%) 326 § 43 196 § 11 300 § 46

Se(VI) reduced (¹mol) 250 § 19 105 § 34 165 § 69

Se(IV) produced (¹mol) 1003 § 155 703 § 164 1099 § 66

Electrons producedc (¹mol)

822 § 185 624 § 85 1009 § 149

Electrons consumedd (¹mol)

81 § 6 92 § 25 92 § 15

Electrons consumed/ produced (%)

a Experiments were done in triplicate with no-substrate controls to correct for selenate reduction due to metabolism of background carbon introduced with the sediment inoculum. b The increase in cell carbon was estimated to be equal to the increase in protein concentration (Luria 1960) and corrected for background cultures with no substrate. c Calculated on the basis of the following stoichiometric equations: C7 H6 O2 C 12 H2 O ! 7 CO2 C 30 HC C 30 e¡ , or C7 H6 O3 C 11 H2 O ! 7 CO2 C 28 HC C 28 e¡ , and corrected for substrate assimilation to cell mass. 2¡ 2¡ d ¡ ¡ ¡ 0 ¡ Calculated on the basis of the reduction of electron acceptor as follows: SeO2¡ 4 C H2 O C 2 e ! SeO3 C 2OH and SeO3 C 3 H2 O C 4 e ! Se C 6 OH . 0 Se(VI) reduction to Se(IV) (18 ¹mol) in background cultures subtracted from these values. Se concentration calculated as the difference between Se(VI) reduced and Se(IV) produced.

Benzoate 3-Hydroxybenzoate 4-Hydroxybenzoate

Substrate

Substrate useda (¹mol)

TABLE 1 Balance of electrons and acceptors by enrichment cultures initiated with 10% sediment inoculum of Kesterson Reservoir sediment

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The electron balance for substrate oxidation and electron acceptor reduction was determined to establish whether the degradation of benzoate, 3-hydroxybenzoate , and 4-hydroxybenzoat e was coupled to the reduction of Se(VI). Enrichment cultures were fed 700 to 1,000 ¹M substrate over a 77-day period and the reduction of Se(VI), production of Se(IV), and loss of substrate were monitored. The balance of electrons donated and electrons consumed is shown in Table 1. The ratio of electron consumed to electrons produced was 81 to 92% for degradation of the benzoic acid derivatives. Approximately 7 to 10% of the substrate carbon was assimilated to cell mass. These results indicate that anaerobic degradation of the benzoic acid derivatives was dependent on selenate and degradation of each substrate was stoichiometrically coupled to selenate reduction. Isolation of Se(VI)-Reducing Bacteria Selenate-reducing bacteria were independently isolated on 4-hydroxybenzoat e from both Arthur Kill and Kesterson Reservoir and designated AK4OH1 and Ke4OH1, respectively. In soft agar (0.4 % agar) shake tubes with 1 mM 4-hydroxybenzoat e and 10 mM Se(VI), the strains formed bright red colonies (2– 3 mm in diameter) within 5 to 7 days. Degradation of substrate, reduction of Se(VI), and production of Se(IV) by these isolates are shown in Figure 3. Each strain degraded approximately 1 mM 4-hydroxybenzoat e (Figure 3A)

FIGURE 3 Degradation of 4-hydroxybenzoat e (A) and reduction of selenate (B) by strains AK4OH1 and Ke4OH1. Symbols: (A) AK4OH1 H, Ke4OH1 ; (B) Se(VI) AK4OH1 H, and Ke4OH1 , Se(IV) AK4OH1 r, and Ke4OH1 ". Data points are the means of duplicate cultures.

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and reduced 10 mM Selenate (Figure 3B) within 12 days. It is interesting to note that a stoichiometric amount of Se(IV) was produced from Se(VI); therefore, they did not reduce it in substantial amounts beyond Se(IV). The enrichment cultures were capable of reducing Se(VI) to Se0 as demonstrated in the earlier experiments and by the presence of a red precipitate on the colonies in the shake tubes. Experiments to demonstrate conclusively whether the strains are capable of respiring with selenate could not be performed due to the difŽ culty in maintaining the cultures. 16S rRNA gene sequence analysis of the 4-hydroxybenzoat e utilizing strains AK4OH1 and Ke4OH1 isolated from the Arthur Kill intertidal strait and the Kesterson Reservoir sediments placed them within the gamma subgroup of the Proteobacteria (Figure 4). The 16S rDNA gene sequences of strains AK4OH1 (AF432145) and Ke4OH1 (AF432146 ) were 98.3% similar, indicating that the strains were closely related even though they were isolated from very different environments. It is unlikely that enrichment for selenate-reducing bacteria had occurred in the Arthur Kill estuarine sediment prior to establishment of the enrichment cultures. A perchlorate-reducing bacterium, strain NSS (Coates et al. 1999), was their closest neighbor (98.0% similarity to strain AK4OH1 and 97.7% similarity to strain Ke4OH1). The use of other electron acceptors by strain NSS was not reported. Interestingly, the selenate-reducing strains from the Arthur Kill intertidal strait and the Kesterson Reservoir and the chlorate-reducing isolate clustered within a group of uncultured sulfur-oxidizing symbionts from bivalves. It will be interesting to examine the shared physiological properties of the organisms within this clade, in particular their ability to use different electrons donors and acceptors. Several dissimilatory Se(VI)-reducing organisms have previously been isolated in pure culture. These organisms are phylogenetically diverse and, based upon 16S rRNA sequences, have representatives that are afŽ liated with the ¯ (Macy et al. 1993), ° (Knight and Blakemore 1998, this study), and " (Oremland et al. 1994) subdivisions of the

FIGURE 4 Phylogenetic tree showing relationship of Se(VI)-reducing isolates AK4OH1 (AF432145) and Ke4OH1 (AF432146 ) within the Proteobacteria. The tree was constructed from 1116 bp unambiguously aligned bases using fastDNAml. Bootstrap values (