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Applied and Environmental Microbiology, October 2001, p. 4796-4804, Vol. 67, No. 10
Abteilung Mikrobiologie, Fachbereich
Biologie/Chemie, Universität Osnabrück, 49069 Osnabrück, Germany
Received 20 March 2001/Accepted 16 July 2001
Deuterated styrene ([2H8]styrene) was
used as a tracer in combination with phospholipid fatty acid (PLFA)
analysis for characterization of styrene-degrading microbial
populations of biofilters used for treatment of waste gases. Deuterated
fatty acids were detected and quantified by gas chromatography-mass
spectrometry. The method was evaluated with pure cultures of
styrene-degrading bacteria and defined mixed cultures of styrene
degraders and non-styrene-degrading organisms. Incubation of styrene
degraders for 3 days with [2H8]styrene led to
fatty acids consisting of up to 90% deuterated molecules.
Mixed-culture experiments showed that specific labeling of
styrene-degrading strains and only weak labeling of fatty acids of
non-styrene-degrading organisms occurred after incubation with [2H8]styrene for up to 7 days. Analysis of
actively degrading filter material from an experimental biofilter and a
full-scale biofilter by this method showed that there were differences
in the patterns of labeled fatty acids. For the experimental biofilter
the fatty acids with largest amounts of labeled molecules were palmitic acid (16:0), 9,10-methylenehexadecanoic acid (17:0 cyclo9-10), and
vaccenic acid (18:1 cis11). These lipid markers
indicated that styrene was degraded by organisms with a
Pseudomonas-like fatty acid profile. In contrast, the
most intensively labeled fatty acids of the full-scale biofilter sample
were palmitic acid and cis-11-hexadecenoic acid (16:1
cis11), indicating that an unknown styrene-degrading
taxon was present. Iso-, anteiso-, and 10-methyl-branched fatty acids
showed no or weak labeling. Therefore, we found no indication that
styrene was degraded by organisms with methyl-branched fatty fatty
acids, such as Xanthomonas, Bacillus, Streptomyces, or Gordonia spp.
Extraction and analysis of
chemotaxonomically important lipid markers from environmental samples
constitute a well-established method for characterizing microbial
communities, detecting community changes through time, or obtaining
information about the metabolic status of a community
(39). Recently, phospholipid fatty acid (PLFA) analyses
were combined with carbon isotope labeling techniques to link
degradation activities with specific microbial populations (2,
12, 25). 14C tracers were successfully
used for this approach (28). However, the main
disadvantage of the procedure was the low efficiency of separation of
the radiolabeled fatty acid methyl esters (FAMEs) caused by the
discontinuous collection of fractions prior to scintillation counting.
This was avoided by using substrates labeled with the stable
13C isotope, which facilitated continuous
detection of FAMEs by gas chromatography and on-line-combustion isotope
ratio mass spectrometry (27).
We studied the use of a deuterated substrate as an alternative to
13C isotopes for characterization of actively
degrading microbial populations in complex communities. The use of
deuterated substrates with subsequent gas chromatography-mass
spectrometry analysis of the products is a well-established method for
studying metabolic pathways in humans (7, 32). Compared
with 13C-labeled substrates, deuterated compounds
have several advantages: a large number of such compounds are
available, they are less expensive, and the relatively low natural
background of deuterium is beneficial for using this isotope in
isotope-labeling techniques.
In this study, deuterated styrene was used for characterization of
styrene-degrading guilds with labeled fatty acids in PLFA profiles of
biofilters used for treatment of styrene-containing waste gases.
Incorporation of deuterium into fatty acids was detected and quantified
by a gas chromatography-mass spectrometry system. The method was
evaluated by analyzing pure cultures of styrene degraders. Nonspecific
labeling of non-styrene-degrading organisms was quantified in defined
mixed-culture experiments.
Strains.
For pure-culture experiments, the styrene-degrading
organisms Gordonia sp. strain D7 and Pseudomonas
sp. strain D26 were used. These strains were previously isolated from
biofilters that were supplied with styrene, and they were identified by
chemotaxonomic methods. They were deposited in the Deutsche Sammlung
für Mikroorganismen und Zellkulturen, Braunschweig, Germany,
under accession numbers DSM 44441 (Gordonia sp. strain D7)
and DSM 13957 (Pseudomonas sp. strain D26).
Pseudomonas pseudoalcaligenes DSM
50188T and Gordonia terrae DSM
43249T were used as non-styrene-degrading
reference strains. Strains of the genera Pseudomonas and
Gordonia were chosen because they can be clearly
differentiated from each other by means of their fatty acid profiles.
The genus Pseudomonas is characterized by hydroxy fatty
acids, 16:1 cis9, 18:1 cis11, and 19:0 cyclo11-12 (36). In contrast, members of the genus
Gordonia contain the fatty acids 16:1 cis10, 18:1
cis9, and 18:0 10methyl (20).
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4796-4804.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Stable-Isotope-Based Labeling of Styrene-Degrading
Microorganisms in Biofilters
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Defined-culture experiments. Gordonia sp. strain D7 and Pseudomonas sp. strain D26 were cultivated in 500-ml screw-cap flasks containing 150 ml of basal medium and 20 µl of styrene (Merck, Darmstadt, Germany) for 7 days. After the cultures were established, they were each supplemented with 20 µl of [2H8]styrene (Cambridge Isotope Laboratories, Andover, Mass.) and incubated for an additional 3 days. Both strains were also cultivated exclusively with [2H8]styrene or styrene for preparation and analysis of maximum labeled FAMEs.
To determine the amount of the deuterium label transferred from styrene-degrading strains to non-styrene-degrading strains, we performed mixed-culture experiments. For these experiments 10 ml of a Gordonia sp. strain D7 culture grown on styrene was mixed with a 40-ml culture of P. pseudoalcaligenes DSM 50188T which was grown in basal medium supplemented with 0.2% sodium lactate. This mixture was used for inoculation of 100 ml of basal medium. Twenty microliters of styrene was added to each culture, and the cultures were incubated in 500-ml screw-cap flasks. After 7 days, 20 µl of [2H8]styrene was added to each culture, and the cultures were incubated for an additional 3 or 7 days. Mixed cultures of Pseudomonas sp. strain D26 and G. terrae DSM 43249T were also prepared. These cultures were incubated for 3 days, and then 20 µl of [2H8]styrene was added to each culture. The cultures were incubated for an additional 3 or 6 days.Labeling of filter material. Filter material from a styrene-degrading experimental biofilter was kindly supplied by H.-J. Warnecke, Universität-Gesamthochschule Paderborn, Paderborn, Germany. The filter material consisted of a mixture of crushed wood and bark compost and had a water content of 74% (determined by drying at 80°C) and a pH of 4.1 (determined after stirring in 1 M KCl). A filter material sample from a full-scale biofilter was kindly supplied by R. Hübner, Braunschweiger Umwelt-Biotechnologie GmbH, Braunschweig, Germany. This filter was used for treatment of styrene-containing waste gas emitted from a varnishing process. Tree bark compost was used as the filter material. The filter sample had a water content of 69% and a pH of 5.2. For labeling, 10-g portions of biofilter material were incubated in 500-ml screw-cap flasks after addition of 20 µl of [2H8]styrene for 3 to 10 days. Five-gram portions of these samples were used for a PLFA analysis.
Fatty acid analyses. The cells in liquid cultures were harvested by centrifugation. Saponification with 15% NaOH in 50% methanol, acid methylation with 6 N HCl in 50% methanol, and extraction of FAMEs were performed as described by Sasser (29). Lipids in the biofilter samples were extracted by a modified Bligh-Dyer procedure. The lipid extracts were fractionated on silica columns and methylated to FAMEs by mild alkaline methanolysis as described previously (41). The influence of the extraction procedure on the deuterated fractions of the fatty acids was investigated by extracting pure cultures with both methods. The FAME extracts were analyzed by gas chromatography-mass spectrometry with a Hewlett-Packard model 5890 series II gas chromatograph equipped with a 5% phenyl methyl silicone capillary column and a model 5972 mass selective detector as described previously (21). The positions of double bonds and cyclopropyl groups were verified by analyzing the dimethyl disulfide adducts and the dimethyloxazoline derivatives of the FAMEs (24, 44). The positions of hydroxy, methyl, and cyclopropene groups and double bonds were determined from the carboxyl group of the fatty acid molecule according to the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (13).
Quantification of labeled FAMEs.
The amount of a labeled
(deuterated) FAME was calculated as a percentage of the labeled
molecules based on the total amount of molecules of the FAME.
All calculations were performed with averages of the mass spectra from
the whole area of each fatty acid peak. For saturated fatty acids, the
calculation was based on the abundance of the unlabeled molecular ion
and the isotopically modified molecular ions (isotopomeres). The
abundances of all isotopomeres were added after subtraction of
the part of the isotopomeres which resulted from the natural occurrence
of 2H, 13C, and
18O according to the equation:
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(1) |
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(2) |
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Characteristics of deuterated fatty acids.
Incorporation of
deuterium into fatty acids of Gordonia sp. strain D7 and
Pseudomonas sp. strain D26 was detected by the occurrence of
specific isotopomeres in the mass spectra after incubation with
[2H8]styrene. As an
example, the mass spectra of deuterated and unlabeled tuberculostearic
acid (18:0 10methyl) are shown in Fig. 1.
The molecular ion m/z 312 was replaced after growth on
[2H8]styrene by a set of
isotopomeres ranging from m/z 313 to 321 (maximum abundance,
m/z 317), which indicated that on average five atoms of
2H were incorporated per tuberculostearic acid
molecule. The average incorporation of 2H was
between 14 and 26% of all hydrogen atoms for the fatty acids analyzed.
Therefore, the mass spectra of all fatty acids from the reference
strains after growth on
[2H8]styrene could be
clearly differentiated from the mass spectra of unlabeled fatty acids.
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Labeling of pure and mixed cultures.
The labeling experiments
showed that deuterated styrene was assimilated by established cultures
and the assimilation products could be detected by mass spectrometry.
Quantification of labeled fatty acids by using the algorithms described
above demonstrated that the label was not incorporated equally in all
fatty acids (Fig. 3). After 3 days of
incubation with
[2H8]styrene,
Pseudomonas sp. strain D26 showed lower incorporation of
2H in the hydroxy fatty acids 10:0 3OH, 12:0 2OH,
and 12:0 3OH and the cyclopropane fatty acids 17:0 cyclo9-10 and
19:0 cyclo11-12 (27 to 63%) than in the monoenoic and saturated
fatty acids (66 to 89%) (Fig. 3A and B). Gordonia sp.
strain D7 exhibited more homogeneous labeling of fatty acids, ranging
from 41% for 16:1 cis10 to 82% for 18:0 (Fig. 3C and D). A
comparison of FAME extracts prepared by acid methylation (Fig. 3A and
C) with FAME extracts prepared by mild alkaline methanolyis (Fig. 3B
and D) revealed similar contents of label, although the two methods
resulted in differences in the fatty acid profiles (e.g., absence of
hydroxy fatty acids in the PLFA profile).
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Labeling of biofilter material.
Analyses of the filter samples
after incubation with
[2H8]styrene for 3 days
resulted in detection of the 2H marker in a
limited number of the fatty acids detected. The first indications of
specific incorporation of deuterium were nonsymmetrical shapes of fatty
acid peaks. For the experimental biofilter, the most intensely labeled
fatty acids were 17:0 cyclo9-10 (28% labeling) and 18:1
cis11 (for which the labeled fraction consisted of 15% of
the molecules) (Fig. 6). Minor amounts of deuterated fatty acids were found for 16:1 cis9 (9%), 16:1
cis11 (4%), 16:0 (10%), 18:1 cis9 (8%), and
19:0 cyclo11-12 (9%). No labeled molecules were found for linoleic
acid (18:2 cis9,12), octadecanoic acid (18:0), and
arachidonic acid (20:4 cis5,8,11,14). For fatty acids that
accounted for less than 1.5% of the whole profile, quantification of
the labeled molecules was not reliable since the signal-to-noise ratio
was too low for detection of mass fragments or molecular ions of the
isotopomeres. For these fatty acids, labeled molecules were not
quantified. A qualitative evaluation of the mass spectra of these fatty
acids gave no indication of the presence of deuterated fragments or
molecular ions.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The effects of deuterated compounds on enzyme activities are generally considered to be insignificant. This is the basis for extensive use of this isotope as a tracer in human biomedical experimentation and diagnostics (32). The high tolerance of bacteria to this isotope was impressively demonstrated by Vanatalu et al. (35), who prepared completely deuterated ribosomes from a strain of Escherichia coli which were fully active. Our labeling experiments, performed with defined cultures, demonstrated that styrene-degrading isolates grow well even with [2H8]styrene as the only source of carbon. Therefore, use of the deuterated tracer should not have resulted in significant inhibition effects on styrene-degrading populations in the samples analyzed. Thus, clear labeling effects were detected in all samples.
However, incorporation of deuterium affected the chromatographic properties of the FAMEs analyzed. Our analyses showed a strong correlation between the deuteration rate and a decrease in retention time. This chromatographic effect has been described previously for deuterated isotopomeres of several other molecules, including caffeine (5), n-alkanes (23), and fatty acid pentafluorobenzyl esters (26). This isotope effect is primarily attributed to the shorter C---D covalent bond (instead of the longer C---H bond), which modifies several physical properties of the deuterated FAMEs, such as hydrophobicity, which in turn affect the chromatographic properties of the molecules. Partial separation of the unlabeled part of fatty acids from the deuterated part can be used for preliminary detection of labeled fatty acids by their nonsymmetrical peak shapes without interpretation of the mass spectra (Fig. 2A and E). Moreover, the chromatographic separation allows identification of unknown fatty acids based on the late-eluting parts of the peaks. These peak areas exhibit low levels of isotopomere background and can be used for identification of the compounds by mass spectrum libraries. However, the chromatographic shift can cause problems, if the labeled part of a fatty acid overlaps an unlabeled fatty acid with similar retention time and identical mass spectrum, as observed in mixed-culture experiments (Fig. 4 and 5).
The labeling experiments performed with pure cultures of styrene-degrading isolates showed that there was unequal distribution of the deuterium tracer in the fatty acid profiles. For both isolates, incorporation rates can be related to the fatty acid synthesis pathways. For example, the monounsaturated fatty acids of Pseudomonas sp. strain D26, 16:1 cis9 and 18:1 cis11, had a higher labeling rate than the cyclopropyl fatty acids 17:0 cyclo9-10 and 19:0 cyclo11-12 if the culture was incubated for 3 days with [2H8]styrene (Fig. 3 and 4A). This observation is in accordance with the finding that monounsaturated fatty acids are precursors for the synthesis of cyclopropyl fatty acids (18). Thus, the labeling rate of cyclopropyl fatty acids increased when the culture was incubated for 6 days in the presence of [2H8]styrene (Fig. 4B). A corresponding relationship was found for Gordonia sp. strain D7, in which the monounsaturated fatty acids had higher labeling rates than 18:0 10methyl, which is a methylation product of 18:1 cis9 (19). The labeling rate of 18:0 10methyl could be increased by increasing the incubation time with [2H8]styrene from 3 to 7 days (Fig. 5).
Long incubation times of several days with stable isotope tracers could result in nonspecific labeling of other populations in the community. This labeling could have been caused by excretion of secondary metabolites by the primary degrading population, which were assimilated by other microorganisms. Moreover, deuterium ions could be produced by dehydrogenation reactions or formation of carboxyl groups during degradation of the 2H tracer. This could result in intra- and extracellular enrichment of deuterium ions in the sample and subsequent nonspecific incorporation by other organisms. The loss of covalently bound deuterium from [2H8]styrene during degradation and fatty acid synthesis was indicated by the average incorporation of only five to eight deuterium atoms per fatty acid molecule, which corresponded to 14 to 26% of the hydrogen atoms of the molecule. This shows that the majority of the deuterium was replaced by [1H] and was released into the hydrogen ion pool of the sample. However, in our mixed-culture experiments, we showed that this effect did not result in labeling of non-styrene-degrading strains. Characteristic fatty acids of the non-styrene-degrading organisms used, like 18:0 10methyl (Fig. 4) and 18:1 cis11 (Fig. 5) had no label, even when the incubation time with [2H8]styrene was extended from 3 to 6 or 7 days.
Starting from these data, we were able to show the efficiency of this technique for analysis of biofilter material. For both of the samples analyzed, the complex fatty acid profiles detected indicated the presence of diverse microbial communities (Fig. 6 and 7). Iso- and anteiso-branched-chain fatty acids accounted for a 6.3% portion for the experimental biofilter and a 11.0% portion for the full-scale biofilter (Fig. 7). These lipids are characteristic compounds of bacterial taxa like the Microbacteriaceae, the Streptomycetaceae, the Bacillus-Staphylococcus group, the Xanthomonas branch of the Proteobacteria, and the Bacteroides-Cytophaga phylum (14). For both samples, the major portion of the fatty acids consisted of straight-chain fatty acids, including unsaturated and cyclopropyl fatty acids. Within these fatty acids the products of the aerobic and anaerobic synthesis pathways have been detected. The aerobic synthesis pathway, which results in production of 18:1 cis9 and 18:2 cis9,12, is characteristic of microeukaryotes, the Actinobacteria, and some families in the Proteobacteria, like the Pasteurellaceae and the Moraxellaceae (9, 16, 31, 39). The anaerobic fatty acid synthesis pathway, with the characteristic products 18:1 cis11 and 19:0 cyclo11-12, is expressed by most members of the Proteobacteria. Both samples were characterized by large amounts of 18:2 cis9,12 and 18:1 cis9, which indicates that major quantities of microorganisms with the aerobic fatty acid synthesis pathway were present in both biofilters.
For both samples, the styrene-degrading populations could be characterized by detection of a subset of deuterated fatty acids. For the experimental biofilter the highly labeled (>7%) fatty acids were 16:1 cis9, 16:0, 17:0 cyclo9-10, 18:1 cis9, 18:1 cis11, and 19:0 cyclo11-12. These compounds are the dominant fatty acids of phospholipid fractions of the genus Pseudomonas (36). The results are in accordance with the previous isolation of a number of styrene-degrading strains of the genus Pseudomonas from this experimental biofilter (22), one of which, Pseudomonas sp. strain D26, was chosen for the pure- and mixed-culture studies reported here. Thus, the subset of highly labeled fatty acids of the biofilter sample corresponded to the labeled fatty acids of the PLFA profile of Pseudomonas sp. strain D26 shown in Fig. 3B. The data indicated that microorganisms with Pseudomonas-like fatty acid profiles represented the primary degrading population in the experimental biofilter.
Compared to the experimental biofilter, the full-scale filter had a higher diversity of labeled fatty acids. Major labeled portions were found for the fatty acids 16:1 cis11, 17:0 cyclo9-10, 17:0 cyclo11-12, 16:0, 18:2 cis9,12, and 18:1 cis11. In contrast to the experimental biofilter sample, the fatty acid 18:2 cis9,12 was significantly labeled in this sample. This implies that at least one additional microbial group was involved in the assimilation of styrene. The possible candidates include microeukaryotes and some bacterial taxa, such as members of the Pasteurellaceae, in which this fatty acid is found (31). A further remarkable result was the large amount and strong labeling of the fatty acid 16:1 cis11. The intensity of the label suggests that this lipid represented a microbial population in the full-scale biofilter with high styrene-degrading activity. This fatty acid is an unusual compound in microbial fatty acid profiles since the aerobic and anaerobic fatty acid synthesis pathways lead to preferential production of the cis9 isomere of palmitoleic acid (30). Examples of 16:1 cis11-producing taxa are the type I methylotrophic bacteria (4), the family Sphingomonadaceae (34), and the genus Nitrospira (Lipski, unpublished data). However, the absence of labeling or the low labeling rate for other characteristic fatty acids of these taxa argues against participation in the styrene-degrading process of the full-scale biofilter. The characteristic fatty acids are 16:1 cis10 for the type I methylotrophs, 15:0 iso and 17:1 iso cis9 for the Sphingomonadaceae, and 16:1 cis7 for the genus Nitrospira. Moreover, the occurrence of styrene-degrading strains in the methylotrophic bacteria and in the genus Nitrospira is unlikely due to their specialized physiology. The former is characterized by obligate assimilation of C1 substrates (4), and the latter is characterized by obligate chemolithotrophy (8, 42). Therefore, the strong incorporation of deuterium from styrene in 16:1 cis11 indicated that the identity of an important styrene-degrading organism in the full-scale biofilter is still unknown. The characteristic lipid marker 16:1 cis11 could be used to evaluate the importance of styrene-degrading isolates from this biofilter in future enrichment studies.
Fatty acids that are not present or not labeled can also provide useful information about the community in biofilter samples. For example, we did not detect 18:0 10methyl in the experimental biofilter or the full-scale filter, which indicates the minor quantitative importance of genera like Gordonia, Nocardia, Mycobacterium, Aeromicrobium, Nocardiopsis, and Actinomadura, all of which are characterized by major amounts of 18:0 10methyl (3, 11, 15, 17, 40, 43). Characteristic fatty acids which were present but not labeled in the experimental biofilter were the iso- and anteiso-branched-chain fatty acids and the polyunsaturated fatty acids. This indicates the presence of several groups of microorganisms which were not involved in the assimilation of styrene. This is of particular importance because some of these groups are known for their styrene-degrading potential. Arnold et al. (1) isolated a Xanthomonas-like styrene-degrading strain from a laboratory-scale peat biofilter. The Xanthomonas branch of the Proteobacteria is characterized by the predominance of iso- and anteiso-branched fatty acids (10, 37). Cox et al. (6) isolated a number of styrene-degrading fungi from several experimental biofilters. Fungi are polyunsaturated fatty acid-containing microorganisms (33). Our analysis clearly eliminated these groups and organisms with similar fatty acid profiles as candidates for the styrene-degrading population in the experimental biofilter analyzed.
Our study showed that 2H tracers can be effectively used in stable-isotope PLFA studies for characterization of actively degrading microbial populations. The chromatographic shifts of deuterated lipid markers require careful analysis of compounds with similar retention times but allow mass spectrometric identification on the basis of the partially separated unlabeled parts of the peaks. In many cases, detection of highly labeled characteristic lipids allows identification of the actively degrading taxa. However, a large data set of lipid profiles is necessary to identify the correct taxon at the appropriate taxonomic rank and to account for the polyphyletic occurrence of many fatty acids. The large amount of available fatty acid data allows identification of constitutive lipid markers, which are synthesized independently, from physical and chemical parameters of the environment.
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ACKNOWLEDGMENTS |
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We thank Karlheinz Altendorf for his support of this study, Udo Friedrich for critical reading of the manuscript, and Ngoc Quynh Lieu for excellent technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Universität Osnabrück, Abteilung Mikrobiologie, Fachbereich Biologie/Chemie, 49069 Osnabrück, Germany. Phone: 0049 541 969 2276. Fax: 0049 541 969 2870. E-mail: Lipski{at}biologie.uni-osnabrueck.de.
Dedicated to Karlheinz Altendorf on the occasion of his 60th birthday.
Present address: FG Ökologie der Mikroorganismen, Technische
Universität Berlin, 10587 Berlin, Germany.
§ Present address: Max-Planck-Institut für terrestrische Mikrobiologie, 35043 Marburg, Germany.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, October 2001, p. 4796-4804, Vol. 67, No. 10
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4796-4804.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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