Environ. Sci. Technol. 2002, 36, 4334-4345
Utilization and Transformation of Aquatic Humic Substances by Autochthonous Microorganisms N . H E R T K O R N , † H . C L A U S , ‡,# PH. SCHMITT-KOPPLIN,† E . M . P E R D U E , § A N D Z . F I L I P * ,‡ GSF-Forschungszentrum fu ¨ r Umwelt und Gesundheit, Institut fu ¨r O ¨ kologische Chemie, Ingolsta¨dter Landstrasse 1, D-85764 Neuherberg, Germany, Umweltbundesamt (Federal Environmental Agency), Langen Building, Paul-Ehrlich-Strasse 29, D-63225 Langen, Germany, and School of Earth and Atmospheric Sciences, Georgia Institute of Technology, Atlanta, Georgia 30332-0340
Aquatic humic substances (HS) from a bog lake water, a river water, and a groundwater were isolated after enrichment on XAD 8 columns and added to a Czapek-Dox nutrient broth which was used either in full strength or without glucose and/or NaNO3. The individual flasks were inoculated with natural microbial populations of corresponding water samples or with a Pseudomonas fluorescens strain isolated from groundwater. The presence of HS resulted in an increase of bacterial numbers in nearly all cultures incubated for 3 weeks at 25 °C on a shaker. HS reisolated from cultures without glucose or NaNO3 showed no or only minor quantitative differences as compared to those from sterile controls. In full strength nutrient broth up to 27% of HS were utilized. Data obtained by spectroscopic methods (UV/vis/FTIR) and elemental analysis indicated a decrease in particle size and a loss in aromaticity and aliphatic carbon in HS reisolated from the microbial cultures. Simultaneously an increase in the N content of HS was observed, which probably originated from some constituents of microbial biomass such as proteins and amino sugars. The NMR data also documented that significant transformations of HS occurred in the individual microbial cultures. After incubation, increased amounts of aromatic acids were detected in some liquid media and residual HS by GC/ MS or capillary electrophoresis. 1H NMR spectroscopy was less effective in indicating structural differences in the HS than 13C NMR but revealed considerable detail of the microbial degradation of riverine HS, when limited sample was available. The newly developed NMR increment analysis provided substantial detail of aromatic structures in a microbially altered HS. The microbial degradation of HS strongly depended on the composition of the HS, the species selection of the microorganisms, and to a lesser extent on the culture conditions. For any series of identical inoculum and HS, full broth media initiated the most extensive alteration of HS. * Corresponding author phone: +49-6103-704-160; fax: +49-6103704-147; e-mail:
[email protected]. † GSF-Forschungszentrum fu ¨ r Umwelt und Gesundheit. ‡ Umweltbundesamt (Federal Environmental Agency). § Georgia Institute of Technology. # Present address: Johannes Gutenberg-Universita ¨ t Mainz, Institut fu ¨r Mikrobiologie und Weinforschung, Becherweg 15, D-55099 Mainz. 4334
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Introduction Humic substances (HS) are the most abundant natural organic constituents in terrestrial and aquatic environments. They represent about 25% of total organic carbon on earth and 50% of organic carbon in oceans and freshwaters (1). Because of their complex chemical structure, it has been disputed whether they are easily decomposable. For aquatic HS, this question has gained special importance, because they have been recognized as precursors of trihalomethanes in chlorinated waters and also as vehicles of metal ions and different organic pollutants (2). For soil HS, a steady-state equilibrium of formation and degradation has been assumed (3). Depending on their origin and other conditions, HS may be readily utilized by microorganisms (4-6). HS from salt marsh environments, e.g., have been shown to undergo microbial transformation and degradation under both aerobic and semi-anaerobic conditions (7). In some surface waters as much as 80% of the dissolved carbon is humic matter (8). Aquatic HS have been shown to affect microbial activities (9, 10) and biomass formation (11-16) and to become degraded in the presence of easily utilizable nutrients (17-21). In our earlier studies we found that some aquatic HS could be preferably utilized as a source of N by authochthonous microorganisms (7, 22). To further elucidate transformation of organic matter occurring in aquatic environments, the bioavailability of HS should be investigated in more detail. Following this goal, the aim of our study was to establish whether HS from groundwater, a river, and a bog lake water can resist microbial degradation or whether they can serve as carbon and/or nitrogen sources for microorganisms indigenous to the aquatic environments.
Materials and Methods Humic Substances. HS were isolated from water samples by standard procedures as described by Abbt-Braun et al. (23). From a bog lake water (Hohlohsee, Germany), they were collected on a XAD-8 [poly(methyl methacrylate)] resin column at pH 2.0 and then eluted with 0.1 M NaOH. Humic acids (HA) were separated from the eluate by precipitation at pH 1.5 and centrifugation (10000g, 15 °C). The supernatant containing fulvic acids (FA) was passed through a cationexchange resin (AG MP 50, H+ form) and freeze-dried. HA from a groundwater aquifer located at Fuhrberg (Germany) were obtained by an acid precipitation as above of HS samples concentrated on polystyrene resins, followed by dialysis against distilled H2O and freeze-drying. The ash content of the final preparations was less than 1.0%. Riverine HS (22) were isolated by ultrafiltration from water samples of the St. Mary’s River (GA, U.S.A.). The fraction of 10-50 kilodaltons was used in the experiments. The ash content was ca. 15%. Microbiological Methods. Water samples from a bog lake and a 140 m deep groundwater well (Langen, Germany) were taken with sterile precautions. Colony forming units (CFU) were counted after 7 days at 25 °C on R2A agar (Difco No. 1826-17-1) for bacteria and on Czapek-Dox agar (Oxoid No. CM 97) for microscopic fungi (standard deviation of CFU counts: 10%). Numbers of bacteria were determined microscopically after fluorescent staining of cells with acridinorange according to Bloem (24). For the approximate determination of bacterial diversity in water samples 125 bacterial isolates obtained from R2A agar were proceeded in the BIOLOG (Hayward, USA) GN and GP microplates and evaluated using a reader and software. HS Utilization Experiments. Freeze-dried HS (HA or FA preparations) from groundwater and lake water were dis10.1021/es010336o CCC: $22.00
2002 American Chemical Society Published on Web 09/14/2002
TABLE 1. Humic Substances, Inocula, and Nutrient Conditions Used in the Degradation Experimentsa
a
humic substance
inoculum
variant
HS no.
lake water FA (Hohlohsee) lake water FA (Hohlohsee) lake water FA (Hohlohsee) lake water FA (Hohlohsee) lake water FA (Hohlohsee) lake water FA (Hohlohsee) lake water FA (Hohlohsee) lake water FA (Hohlohsee) lake water FA (Hohlohsee) lake water FA (Hohlohsee) groundwater HA (Fuhrberg) groundwater HA (Fuhrberg) groundwater HA (Fuhrberg) groundwater HA (Fuhrberg) groundwater HA (Fuhrberg) groundwater HA (Fuhrberg) groundwater HA (Fuhrberg) groundwater HA (Fuhrberg) groundwater HA (Fuhrberg) groundwater HA (Fuhrberg) river water FA (St. Mary) river water FA (St. Mary) river water FA (St. Mary) river water FA (St. Mary) river water FA (St. Mary) river water HA (St. Mary) river water HA (St. Mary) river water HA (St. Mary) river water HA (St. Mary) river water HA (St. Mary) river water HA (St. Mary)
groundwater (Langen) groundwater (Langen) groundwater (Langen) groundwater (Langen) lake water (Hohlohsee) lake water (Hohlohsee) lake water (Hohlohsee) lake water (Hohlohsee) sterile control sterile control groundwater (Langen) groundwater (Langen) groundwater (Langen) groundwater (Langen) lake water lake water lake water lake water sterile control sterile control river water river water river water river water sterile control river water river water river water river water sterile control sterile control
F-C F-N F M F-C F-N F M M M F-C F-N F M F-C F-N F M M M F-C F-N F M M F-C F-N F M F-C M
FA 3 FA 2 FA 1 FA 4 FA 7 FA 6 FA 5 FA 8 FA 9 FA 10 HA 13 HA 12 HA 11 HA 14 HA 17 HA 16 HA 15 HA 18 HA 19 HA 20 FA 24 FA 23 FA 22 FA 25 FA 21 HA 28 HA 27 HA 26 HA 29 HA 30 HA 31
F: full nutrient broth; F-C: C-deficient medium, F-N: N-deficient medium; M: mineral solution.
solved in 0.1 N NaOH, sterilized by membrane filtration (0.2 µm), and added to 100 mL of autoclaved nutrient broth at final concentrations of ca. 1.0 mg mL-1. The final solutions with the riverine HS contained a natural mixture of about 210-260 µg mL-1 of HA and 190-200 µg mL-1 of FA (22). No particulate humic materials could be visualized in the flasks. A Czapek-Dox nutrient broth (composition in g L-1: glucose 30, NaNO3 3.0, K2HPO4 1.0, MgSO4‚7 H2O 0.5, KCl 0.5, FeSO4. 7 H2O 0.01; pH 7.0) was used in full strength (F) or without either glucose and/or NaNO3 to receive a carbon-deficient (F-C), a nitrogen-deficient (F-N), or a “mineral” (M) nutrient solution. The respective inoculum consisted of 1.0 mL of original water and 0.5 mL of a natural culture enriched in PYGV medium after Staley (25). In some experiments a Pseudomonas fluorescens strain isolated from groundwater and enriched in PYGV medium was used as inoculum. The individual flasks were inoculated at a final concentration of about 1.0 ‚ 107 mL-1 CFU and incubated in the dark at 25 °C on a reciprocal shaker for 3 weeks. All experiments were performed in duplicate; the relative uncertainty of CFU count was below 10%. Inoculated flasks without HS and noninoculated sterile solutions containing HS served as controls. Table 1 contains HS, inoculum, and nutrient conditions for the degradation experiments used. At the end of incubations CFU numbers in the individual assays were determined on a R2A agar. Total microbial biomass was harvested by centrifugation, washed repeatedly with distilled H2O, and freeze-dried. Supernatant culture solutions were acidified (pH 1.5), and HA were allowed to precipitate overnight. They were separated by centrifugation, dialyzed against deionized water, freeze-dried, and weighted. FA were isolated from the remaining supernatants on a XAD-8 column, eluted with 0.1 M NaOH, passed through another resin column (AG MP 50, H+ form), and freeze-dried. Analysis of HS. Elemental Analysis. Carbon, hydrogen, and nitrogen contents in HS were determined using a Perkin-
Elmer Analyzer Model 240 C. Oxygen was evaluated by difference. Ash content was determined after heating the samples at 500 °C overnight. UV/vis Spectroscopy. The light absorbance of HS (200 ppm HS redissolved in 0.05 M NaHCO3) was measured over the wavelength range 200-800 nm in a Spectronic Genysis 5 photometer (Milton-Roy, USA). The absorbances at 465 and 665 nm were used for calculation of the E4/E6 ratios (26). Absorbance at 280 nm was used to evaluate aromaticity (27). FTIR Spectroscopy. For Fourier transform infrared spectroscopy (FTIR) studies, KI pellets containing 2% of sample were pressed at 250 atm and examined in a Bruker IFS 85 FTIR spectrophotometer over the wavelength range 2.5-15 µm (4000-300 cm-1). NMR Spectroscopy. 13C NMR spectra were recorded with a Bruker AC 400 NMR spectrometer and a 5 mm broadband probe with a antiring sequence (28) under WALTZ broadband decoupling with full NOE: SW 35714 Hz with 32k data points, corresponding to an acquisition time of 459 ms and a relaxation delay of 1 s; FIDs were zero filled to 64 k and multiplied with a line broadening of 100 Hz, providing a digital resolution of 1.1 Hz/point. The chemical shift was referenced to external CH3OH in D2O: 49.00 ppm and internal HDO: 4.63 ppm. 1H NMR spectra were recorded with a Bruker DMX 500 NMR spectrometer and a 5 mm inverse geometry broadband probe using a NOESY-presat sequence employing 90-pulses (10.8 µs): SW 12531 Hz with 8k data points, corresponding to an acquisition time of 327 ms, a mixing time of 200 ms and a irradiation time of 2 s; FIDs were zero filled to 16 k and multiplied with a line broadening of 0.75 Hz, providing a digital resolution of 1.1 Hz/point. The integration of NMR spectra was accurate to better than 5% referred to the respective areas shown in Tables 6-9; in regions of very low S/N ratio it went up to 15%. 2D NMR spectra of 2 mg of substance HA 26 (Table 1) were recorded with a Bruker DMX 500 spectrometer (proton VOL. 36, NO. 20, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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(gradient pulse: 1 ms, gradient recovery: 450 µs; HSQC: aq: 195 ms, sw: 5252 Hz, d1: 1 s, 1J(CH): 145 Hz, 13C GARP decoupling: 70 µs, number of increments in F1: 88. The FID of the HSQC spectrum was multiplied 30 Hz line broadening in F2 and by an π/2 shifted sine bell in F1 dimension; the spectrum was zero filled providing a 2k ‚ 256 data matrix. Increment analysis was performed with standard values for neutral substituents (29, 30). For anionic hydroxyl (o/ m/p (1H,13C): -0.47, -10.8/-0.36, 1.0/-0.85, 21.5 ppm) and anionic carboxyl (o/m/p (1H,13C): 0.56, 1.3/-0.10, 1.4/0.14, -3.3 ppm), the given values were obtained from linear regression analysis of chemical shift data of simple phenols and carboxylic acids. Capillary Electrophoresis. CE-Instrumentation consisted of a Beckman P/ACE 2050 Series CE (UV-filter used at 254 nm) with the Beckman System Gold Software. Uncoated fused-silica CE capillaries (75 µm id, 375 µm od, 50 cm length to the detector, total length 57 cm) were obtained from Laser 2000 GmbH (Wessling, Germany). Typical capillary zone electrophoresis (CZE) conditions for separation of the various HS fractions were as follows: separation buffer, 50 mM acetate (pH 5.3), 25 mM carbonate (pH 9.3 and adjusted to pH 11.4 with 0.1 NaOH); temperature, 30 °C; voltage, 20 kV; hydrodynamic injection, 10 s as described earlier (31). Dayto-day changes in migration times occurring because of relative changes in the electroosmotic flow (different capillary surface conditions) were controlled by converting the data
TABLE 2. Bacterial Numbers and Species Composition of Water Samples groundwater microscopy 8.6 ‚ 102
lake water
R2A agar
microscopy mL-1
Bacterial Numbers 2.0 ‚ 102 8.7 ‚ 103
R2A agar 8.6 ‚ 103
species identified (no. of positive identifications)
Pseudomonas fluorescens Variovorax paradoxus Xanthomonas campestris Pseudomonas glathei Rhodococcus spec. Cytophaga marinoflava Enterobacter agglomerans Psychrobacter immobiliz Buttiauxella agrestis
9 3 3 2 1 1 1 1 1
Pseudomonas fluorescens Aureobacterium saperdae Escherichia vulneris Rhodococcus spec. Pseudomonas marginalis Comamonas terrigena Enterobacter taylorae Serratia plymuthica Chromobacter violaceum Flavobacterium spec. Pseudomonas putida
15 4 4 4 3 2 2 2 1 1 1
frequency: 500.13 MHz) using an inverse geometry 5 mm probehead (90°: 10.8 µs 1H; 10.0 µs 13C) in 0.1 N NaOD at 303 K (1H/13C: 4.63 ppm/external reference: CH3OH in D2O: 49.00 ppm). Gradient enhanced phase sensitive (TPPI) HSQCspectra were acquired using Bruker standard software
TABLE 3. CFU Counts on R2A Agar after 3 Weeks of Incubationb source of HS/microbial inoculum culture broth
groundwater/groundwater
groundwater/lake water
lake water/groundwater
lake water/lake water
inoculum full broth + HS full broth - HS N-deficient + HS N-deficient - HS C-deficient + HS C-deficient - HS mineral + HS mineral - HS
9.5 ‚ F. The composition of the HA 15 preparation (from full strength broth cultures) was clearly affected by the microbial activity. Here both 1H- and 13C NMR spectra indicated an increase in carbohydrate content and a significant decrease in aromatics. The bog-lake FA recovered from the full strength broth cultures inoculated with the groundwater microflora (FA 1) showed a marked increase in their aliphatic content and slight reductions in aromatic, carboxylic, and keto constituents. The loss of aromaticity correlated with the appearance of aromatic acids in the respective culture broth as detected by GC-MS. Dehorter et al. (46) concluded that humic acid degradation in essence seems not to be a hydrolytic process, in contrast to the degradation of most soil organic polymers. Similar to our experiments, their 13C NMR did not reveal a clear decrease of specific components, suggesting that the more unstable
structures, like carbohydrates, were not attacked initially. However, the 13C NMR spectra presented by Lim and Shin (44) evidenced that the microbial decomposition was concentrated in the more easily available aliphatic fractions of aquatic FA. The decolorization of soil HA by fungi and bacteria has been found to depend both on soil HS structure and on the species selection of microorganisms (47); however, no detailed assessment of the structural changes of HA have been provided in this study. Recently we have shown that rather unspecific acting enzymes such as laccases may be involved in the degradation of aquatic HS (21). In their experiments with Penicillium frequentans, Mathur and Paul (48) found salicyl alcohol as one possible degradation product of soil HS. Some simple aromatic aromatic metabolites, i.e., hydroxy- or methoxybenzoic acids, were detected in our study by GC/MS and CZE analysis. This finding could indicate a microbial cleavage of the complex aromatic core of HS. In general, the combination of microbial population, the structure of HS, and the composition of the culture medium determined the final composition of the reisolated HS. The structure of HS had a significant influence on the course and extent of microbial alteration; e.g. riverine HS, which contained a substantial carbohydrate content, has been altered more thoroughly than the highly aliphatic groundwater and lake water HS. For any series of identical inoculum and HS, full broth media initiated the most extensive alteration (degradation, and possibly resynthesis) of HS, which typically was accompanied by a decrease of aromaticity and an increase in carbohydrate structures.
Acknowledgments The authors wish to thank Ms. M. Schlander (Langen) and Mrs. E. Schindlbeck (Neuherberg) for skilled technical assistance. The late Volker Schinz (Langen) performed the GC/MS analysis. This research was supported by a Grant Fi 268/16-3 and He 2422/1-3 from the Deutsche Forschungsgemeinschaft (DFG) in Bonn as a part of a “ROSIG” Research Program.
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Received for review December 28, 2001. Revised manuscript received July 8, 2002. Accepted July 10, 2002. ES010336O VOL. 36, NO. 20, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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