fingerprinting de bacterias

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Journalof

MethodsMicrobiological

Journal of Microbiological Methods 31 (1998) 99111

Direct fingerprinting of metabolically active bacteria in

environmental samples by substrate specific radiolabelling

and lipid analysis

*Peter Roslev , Niels Iversen, Kaj Henriksen

Environmental Engineering Laboratory, Aalborg University, Sohngaardsholmsvej 57, DK-9000 Aalborg, Denmark

Accepted 1 September 1997

Abstract

Substrate specific radio assays were used for enumeration and fingerprinting of microorganisms in environmental samples.14

Direct fingerprinting was based on incorporation of C-labelled substrates into microbial lipids. A radioactive fingerprint14

was obtained by subsequent radio analysis of whole sample phospholipid ester-linked fatty acids ( C-PLFA fingerprint).14 14

This approach provided a C-PLFA fingerprint of the organism actively metabolizing the added C-labelled substrate.

Labelled and unlabelled PLFAs were analysed as methyl ester derivatives by gasliquid chromatography with flame1 4 1 4

ionization detection. The presence of C-PLFAs were determined by collection of CO produced after combustion of the2

fatty acids. Additional analysis of the microbial community was carried out by analysis of the radioactivity assimilated into1 4 1 4

poly-b-hydroxyalkanoates relative to that assimilated into total phospholipids ( C-PHA/ C-PL ratio). The number of14 1 4

organisms involved in the degradation of a C-labelled substrate was estimated using a C-most-probable-number14 14

technique. These different C-based methods were evaluated by studying [ C]methane oxidation in agricultural soil, and14

[ C]phenanthrene degradation in activated sludge and marine sediment. The radio assays resulted in distinct fingerprints of

the bacterial populations capable of degrading the different radiolabelled substrates. Manipulation of the incubation

conditions (e.g., oxygen status) resulted in significant changes in population specific metabolic activity and labelling pattern.14

Phenotypically related microorganisms appeared to dominate [ C]phenanthrene degradation in activated sludge and marine14

sediment under oxic conditions. Anaerobic [ C]phenanthrene degraders in activated sludge produced a very different14

C-PLFA fingerprint. In methane enriched agricultural soil, aerobic methane oxidation was dominated by organisms most14

similar to the Type I methanotrophic bacteria. Several of the findings obtained by the C-PLFA analysis could not have

been established on the basis of conventional PLFAs analysis alone. The results suggest that variations of this simple14

C-fingerprinting method may be applicable to studies of substrate metabolism in mixed microbial communities. Direct

fingerprinting based on substrate specific radiolabelling may also aid in phenotypic characterization of heterotrophic

microorganisms without the need for enrichment or cultivation. 1998 Elsevier Science B.V.

14 14

Keywords: Radioactive Fingerprinting; Phospholipid ester-linked fatty acids (PLFA); C-MPN; C-PLFA; Phenotypic

Characterization; Active Bacteria; Methane; Phenanthrene

*Corresponding author: Tel.: 145 96 358505; fax: 145 98 142555; e-mail: [email protected]

0167-7012/98/$19.00 1998 Elsevier Science B.V. All rights reserved.

P I I S 0 1 6 7 - 7 0 1 2 ( 9 7 ) 0 0 0 9 4 - 8


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100 P. Roslev et al. / Journal of Microbiological Methods 31 (1998) 99111

1. Introduction organisms involved in the degradation of the added14

C-labelled substrate is obtained by a simple radio

A range of methods has been described for GC analysis of labelled fatty acid methyl esters.

detection and enumeration of microorganisms in

complex microbial communities [13]. These meth-ods include classical enrichment approaches and

techniques based on extraction and analysis of 2. Materials and methods

specific cell components such as nucleic acids and

lipids e.g. [24] . Methods based on extraction and 2.1. Bacteria

fractionation of microbial phospholipid ester-linked

fatty acids (PLFAs) have become increasingly popu- Methylosinus trichosporium OB3b and

lar because of their relative simplicity and good Methylomicrobium album BG8 were obtained from

resolution. PLFAs may be used as sensitive the culture collection at the Department of Biology,

chemotaxonomic biomarkers, and individual fatty University of Warwick, UK. Cultures were grown in

acids or fatty acid signatures can be species or genus a nitrate minimal medium (pH 7) with the following

specific e.g. [48]. In environmental studies, PLFA composition: 10 mM KNO , 3 mM Na HPO , 2 mM3 2 4analysis may be used as a relatively rapid means for KH PO , 0.8 mM Na SO , 0.2 mM MgSO , and

2 4 2 4 4

characterization of microbial communities with re- 0.05 mM CaCl . Trace elements were added after2

spect to both community structure and biomass e.g. autoclaving to give a final concentrations of: 1 mM

[2,7,9,10]. ZnCl , 2 mM CuCl , 1 mM NaBr, 0.5 mM2 2

The majority of the methods that rely on ex- Na MoO , 1 mM MnCl , 1 mM KI, 2 mM H BO , 12 2 2 3 3

traction and analysis of specific cell components will mM CoCl , 1 mM NiCl , and 1 mM EDTA. Iron was2 2

only provide relatively limited information about the added to autoclaved medium as filter-sterilized Fe-

physiological status of the microbial community or EDTA to give a final concentration of 50 mM.3

the metabolic activity of individual cells. This ap- Liquid cultures were grown in 500-cm Erlenmeyer

pears to be the case for many molecular techniques flasks with an initial methane concentration of 5%.

based on the polymerase chain reaction as well asconventional PLFA analyses. Some advances have

2.2. Environmental samplesbeen made in lipid research (e.g., determination of

cyclopropyl and cis/trans fatty acids ratio, and poly-Agricultural soil was collected at Nrre Halne inb-hydroxyalkanoate production), and in use of spe-

Northern Jutland, Denmark. Soil from 510 cmcific nucleic acid markers (e.g., fluorescent 16Sdepth was preincubated at elevated methane con-rRNA probes). However, many of the current meth-

centrations (10 000 ppm) for 30 days to enrich forods are not capable of discriminating clearly betweenmethanotrophic bacteria. The soil samples were usedphysiologically active and less active groups of

14 14for studies of [ C]methane metabolism ( CH , 2.0organisms in situ (e.g., vegetative versus dormant

4

GBq/mmol; Amersham, UK). Activated sludge wasorganisms). As a result, it is often difficult to

collected at Aalborg West sewage treatment plant,attribute a measured biological activity to a specificDenmark. Sludge samples were used in studies ofsubset of the microbial population.

14[ C]phenanthrene metabolism ([benzene ring-U-Here we describe relatively simple methods for14

substrate specific enumeration and direct fingerprint- C]phenanthrene, 4.3 GBq/ mmol; Amersham, UK).

ing of metabolically active bacteria in environmental Coastal marine sediment was collected in Aalborg

samples. The enumeration depends on microbial Harbour, Denmark. Aalborg Harbour receives treated14

production of CO from selected radiolabelled sewage effluent from several sources including the2

14substrates (a C-MPN procedure), whereas the Aalborg West treatment plant (see above). The

fingerprinting technique is based on analysis of sampling site experienced salinities between 12 and

radiolabelled phospholipid ester-linked fatty acids 26 ppt. Marine sediment samples were used in14 14

( C-PLFAs). A radioactive PLFA fingerprint of the studies of [ C]phenanthrene metabolism.


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P. Roslev et al. / Journal of Microbiological Methods 31 (1998) 99111 101

142.3. C-most-probable number (MPN) tration of methane decreased to 0.5 ppm methane

enumeration between additions. Labelling to a specific activity of

approximately 1 kBq/ml culture was accomplished

Enumeration of phenanthrene degrading micro- in 23 days.14

organisms was carried out by a three tube C-most- Methane enriched agricultural soil was incubated14

probable-number technique. The procedure was a with CH to specifically label the bacteria that4

3modification of the method described by Somerville metabolized methane. Soil samples (23 g in 14-cm

14et al. [11]. Environmental samples (10 ml) were serum vials) were spiked with CH twice until a

43

diluted with an inorganic minimal medium (90 ml) total of 1-cm had been consumed (0.2 MBq-23and homogenized in a blender (335 min). The cm ). The initial methane concentration was ad-

inorganic medium had the following composition justed to 1% with unlabelled methane. The samples14

(pH 7): 5 mM NH Cl, 3 mM Na HPO , 2 mM were flushed with air between CH additions to4 2 4 4

14KH PO , 0.8 mM Na SO , 0.2 mM MgSO , 50 mM ensure oxic conditions and to remove CO pro-

2 4 2 4 4 2

CaCl , 10 mM FeEDTA, 0.1 mM ZnCl , 0.2 mM duced by the bacteria. Labelling to a specific activity2 2

CuCl , 0.1 mM NaBr, 0.05 mM Na MoO , 0.1 mM of approximately 30 kBq/ g soil was accomplished in2 2 2

MnCl , 0.1 mM KI, 0.2 mM H BO , 0.1 mM CoCl , 2 3 days.2 3 3 20.1 mM NiCl , and 0.1 mM EDTA. For marine Activated sludge and marine sediment samples

214

samples, the medium was supplemented with 20 g were incubated with [ C]phenanthrene to label21NaCl l . The homogenized samples were diluted microorganisms involved in the degradation of this

serially with the inorganic medium followed by a polycyclic aromatic hydrocarbon (PAH). Water sam-3 3

transfer of 1 ml subsamples to 30-cm serum vials ples (4 ml in 38-cm serum vials) were spiked twice14

containing 1 ml autoclaved water from the sampling with [ C]phenanthrene in hexane until a total of

site. The serum vials contained a 25 mm sterile glass 0.2 MBq had been consumed. The additions14 14

fibre filter on which [ C]phenanthrene had been corresponded to2310 nmol [ C]phenanthrene/ ml

blotted. The filter strips were prepared by transfer- sample. Activated sludge was incubated under both14

ring 5 ml of [ C]phenanthrene in hexane onto the oxic and anoxic denitrifying conditions. Anoxic

filter followed by a 1 h drying period to allow the samples were spiked with 10 mM KNO and then34hexane to evaporate. The initial count was 5310 flushed with N for 30 min to establish anoxia.

21 4

cpm per filter (0.2 nmol [ C]phenanthrene). A 5- Labelling to a specific activity of approximately 253

cm glass vial containing 1 ml 1 M NaOH was kBq/ ml sample was accomplished in 57 days3

placed in the serum vial to trap CO . The 30-cm (depending on the initial degradation activity and2

serum vials were capped with rubber stoppers and oxygen status).

incubated for four weeks in the dark at room

temperature on a shaker (50 rpm). After the incuba- 2.5. Extraction and fractionation of lipids

tion, the CO trap was removed and the radioactivity2

was determined in a 0.5 ml NaOH subsample. Lipids were extracted from bacterial cultures (12

Counts exceeding two times the background (un- mg dry weight) and environmental samples (23 g)

inoculated controls) were scored positive. partly as described by Petersen and Klug [12]. The3samples were incubated in 35-cm Pyrex tubes with

2.4. Radiolabelling of bacteria 3 ml 0.1 M phosphate buffer (pH 7.4), 8 ml

methanol and 4 ml dichloromethane (DCM). After

Pure cultures of the methanotrophic bacteria M. extraction overnight, 3 ml phosphate buffer and 4 ml

trichosporium OB3b and M. album BG8 (100 ml in DCM was added to create phase separation. The3

500-cm Erlenmeyer flasks) were radiolabelled by DCM phase was transferred to a second set of Pyrex14

adding CH to the cultures twice until a total of tubes and the solvent evaporated under a stream of4

3 231-cm had been consumed (0.2 MBq cpm-cm ). N . The precipitate was redissolved in chloroform

2

The initial methane concentration was adjusted to and transferred to SiO columns (IST, Mid-Glamor-

1% with unlabelled methane. Headspace concen- gan, UK).


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The extractable lipids were separated into three samples (A. White, Microbial Insights Inc., TN,

lipid classes by silicic acid column chromatography USA). Tridecanoic acid (13:0) and nonadecanoic

[13,14]. The lipids were eluted with 2 ml chloro- acid (19:0) served as internal standards (200 mM).

form, 6 ml acetone, and 2 ml methanol. The three Routine FAME analysis was carried out using a 70

fractions represent neutral lipids (e.g., steroids, min temperature program: 1 min at 608C, 60 toquinones, di and triglycerides), glycolipids (e.g., 1708C at 308C/min, 170 to 300 at 28C / min. Analysis

glycolipids, poly-b-hydroxyalkanoates), and polar of samples with radiolabelled FAMEs was carried

lipids (e.g., phospholipids) [6,15]. The acetone out using a 150 min temperature program: 1 min at

fraction containing poly-b-hydroxyalkanoate (PHA) 608C, 60 to 1708C at 308C/min, 170 to 2308C at

and the methanol fraction containing phospholipid 0.58C/min, 230 to 3008C at 308C/min, and finally 23

(PL) were used for further analysis. PHAs were min at 3008C. The temperature program was paral-

separated from glycolipids by the filter paper method leled by a column pressure program: 5 min at 200

of Findlay and White [13]. Subsamples of 100 ml kPa, 200 to 50 kPa at 30 kPa/ min, 115 min at 50

from the acetone fraction were transferred to 25 mm kPa, and finally 50 to 200 kPa at 30 kPa/ min. The

Whatmann filter paper and the solvent removed 150 min temperature and pressure program resulted

under a stream of warm air. The PHAs were heat in less distinct and relatively broad peaks but pro-fixed to the filter paper by incubation at 808C for 30 vided separation by up to several minutes of in-

min. The filter paper was then washed twice with 2 dividual FAMEs.14

ml ethanol and twice with 2 ml diethyl ether. The Radiolabelled FAMEs ( C-PLFAMEs) were col-14

radioactivity associated with the PHAs was deter- lected manually as CO after column separation2

mined by direct liquid scintillation counting of the and combustion in the GCFID. The outflow from

filter paper. Radioactivity in the PL fraction was the FID was passed through a CO trap consisting of2

determined by counting of 100 ml subsamples. a scintillation vial with a steel hypodermic needle

mounted in the cap (0.8 mm i.d.). The needle was

submerged in a 10 ml mixture of ethanolamine and

2.6. Analysis of PL-FAMEs ethyleneglycolmonomethylether (1:7 vol/ vol) [16].

The needle tip was modified (perforated) to facilitateThe phospholipid containing polar lipid fraction release of small bubbles. The trapping solution

was dried and then subjected to mild alkaline retained CO (labelled and unlabelled) derived from2

methanolysis [12]. The resulting phospholipid ester- the FAMEs whereas other gases escaped through anlinked fatty acid methyl esters (PL-FAMEs) were opening in the vial cap. Two CO traps were

2

dissolved in hexane and analysed on a Hewlett operated in parallel which allowed switching be-Packard 5890 series II GC equipped with an auto- tween traps without interrupting the column and

sampler, a FID detector, and a HP Ultra 2 capillary detector gas flow. The flow from the GCFID outlet3column (50 m30.2 mm i.d.). Samples (15 ml) were was 440-cm gas / min resulting in a mean transfer

analysed in a splitless mode. The GC injector time of,0.2 sec from the FID to the CO trap. The2

temperature was 2708C, the detector temperature presence of radiolabelled fatty acids in individual

3008C, and the initial column temperature was 608C. fractions was detected after scintillation counting.3H was used as a carrier (1.6 cm / min initially), N Only fractions exceeding 23the background radioac-

2 23

as make up gas (35 cm / min), H and air for the tivity were scored positive (2330 cpm for non-2

3flame ionization detector (34 and 370 cm / min, radioactive controls). Radiolabelled PL-FAMEs were

respectively). collected in 15 fractions according to their equivalent

Individual fatty acids were identified tentatively chain lengths (Table 1).

based on retention times relative to authentic stan- Control experiments showed that the total amount

dard fatty acid methyl esters (Nu Chek Prep Inc., of radioactivity recovered from triplicate samples

MN, USA). The identity of selected fatty acids was may vary slightly but the relative distribution of

resolved further by GCMS analysis of parallel activity in individual fractions (%) was always


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14Table 1 tion of C-labelled substrates to intact environmen-Sampling scheme and equivalent chain lengths (ECL) for radio- tal samples was used to specifically radiolabel micro-labelled PLFAs

organisms involved in the degradation of the added14Fraction Time (min) ECL compound (Fig. 1). During the degradation, C

derived from the substrate was assimilated into1 045 ,13.62 4560 13.614.6 microbial biomass which resulted in labelling of3 6070 14.615.3 various macromolecules including different lipid4 7075 15.315.7

classes (e.g., poly-b-hydroxyalkanoates and phos-5 7580 15.716.1

pholipids). Subsequently, a relative simple radio GC6 8085 16.116.5analysis of whole sample PLFAs made it possible to7 8590 16.516.8

148 9095 16.817.1 discriminate between C-PLFAs produced by organ-9 95100 17.117.4 isms involved in degradation of the labelled substrate

10 100105 17.417.7and non-labelled PLFAs from other microorganisms.

11 105110 17.717.9Approximately 15% of the radioactivity incorpo-12 110115 17.918.4rated by the microorganisms in environmental sam-13 115120 18.418.8

14

14 120125 18.819.0 ples was recovered as C-PLFAs. Physiologically15 125150 .19.0 active bacteria with the ability to utilize the added14

C-substrate (and labelled metabolites) as carbon14

source was detected by the described C-PLFA

analysis.comparable (SE,5%). Results presented here repre-

Radiolabelled PLFAs were separated by capillarysent profiles for homogenized samples labelled and14

GC and collected as CO (15 fractions) afteranalyzed in duplicates.2

combustion in the flame ionization detector. Each14

fraction was collected manually and the CO2.7. Liquid scintillation counting2

activity was determined by liquid scintillation count-

ing (LSC). The recovery of radiolabelled PLFAs asThe radioactivity in CO traps used for GC2

1414

CO was 72% (64% SE) of the activity measuredanalysis of C-PLFAs was determined using 12 ml 2 14Packard Instagel Plus as scintillation cocktail. The by direct LSC of the C-PLFAs (collection ef-

radioactivity associated with alkaline liquid samples ficiency). The principles of such radio GC techniques14

( C-MPN), and dry filter paper (PHA) was de- have been described previously e.g. [1618]. An

termined using 15 ml Packard Instagel Plus and outline of the collection scheme used for sampling of

Packard Ultima Gold F, respectively. All samples radiolabelled fatty acids is shown in Table 1. The

were counted for 5 min in a Packard 1600 TR liquid corresponding GC temperature and pressure program

scintillation counter. The counts were corrected for was designed to give sufficient FAME separation14

quench using internal and external standards. necessary for manual collection of CO and to2

provide maximum resolution of C to C fatty14 19

acids. The GC program did broaden individual peaks

3. Results and discussion which led to occasional tailing of some FAMEs. Inthis context, it should be noted that radiolabelled

143.1. C-PLFA analysis FAMEs may have slightly wider peaks than non-

14labelled PLFAs due to the presence of both C and12

A combination of different radio assays were used C atoms [18]. Routine fingerprinting was carried

to characterize and enumerate metabolically distinct out with time intervals down to 5 min. However,

groups of bacteria in environmental samples (Fig. 1). further fractionation with 1 min time intervals was

Substrate specific enumeration of microorganisms also possible. Fatty acids with retention times sepa-

was carried out using serially diluted samples and a rated by less than 1 min were not always resolved14

modified C-most probable number approach. Addi- well by the chromatographic procedure.


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Fig. 1. Outline of the scheme used for specific labelling and analysis of microorganisms in environmental samples. The abundance of14 14

microorganisms capable of degrading the added C-substrate was determined by a three tube C-most-probable-number approach14 14

( C-MPN); the organisms actively metabolizing the C-substrate were characterized by analysis of the radioactivity associated with PLFAs14 14 14

( C-PLFA fingerprint). Additional phenotypic information was obtained from determination of the C-PHA/ C-PL ratio (see text for

details).

14The signature obtained by radio GC analysis of 3.2. Fingerprinting of [ C]methane oxidizers

labelled PLFAs corresponded well with the signatureobtained by standard GC analysis of total PLFAs The monounsaturated phospholipid fatty acids(Fig. 2). A pure culture of the methanotroph M. 16:1v8c and 18:1v8c have been proposed as specifictrichosporium OB3b was grown on CH to early biomarkers for certain Type I and Type II methanot-

414

exponential phase and then labelled with CH . The rophic bacteria, respectively [810,20,21]. Type I4PLFA composition was determined by conventional methanotrophs Methylomonas and Methylococcus

GCFID analysis followed by quantification of the may produce 16:1v8c whereas the Type II methano-14 14

C-PLFAs as CO . The results suggest that the trophs Methylosinus and Methylocystis may produce2

14detection method for C-PLFAs did not create a 18:1v8 [8,20,22].

bias towards certain fatty acids and likely reflects the An agricultural soil was enriched for 1 month at

overall PLFA composition of bacteria growing ac- 1% CH and then analyzed by a conventional PLFA4

14 14tively. However, some differences in isotopic com- approach and after radiolabelling with CH ( C-

4

position of individual lipid classes may be found PLFA analysis). The methane enrichment resulted in

during changes in growth stage for single organisms a significant increase in the abundance of the fatty

[19]. acids 16:1v8c and 18:1v8c (Table 2). The con-


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Fig. 2. Phospholipid fatty acid composition of M. trichosporium

OB3b determined by conventional GC-FID detection (weight %14

PL-FAMEs), and by analysis of C-PLFAs extracted from

radiolabelled samples. The chromatographic run (150 min) wasdivided into 15 fractions as described in Materials and Methods.

Time intervals and the corresponding Equivalent Chain Length

values (ECL) are shown in Table 1.

centration of these PLFAs was below the detection

limit in samples taken before the enrichment. The

presence of 16:1v8c and 18:1v8c after methane

enrichment suggests significant growth of both Type

I and II methanotrophic bacteria in the soil. Radio-14

labelling of the same soil with CH followed by a4

14

1 4C-PLFA analysis resulted in important additional Fig. 3. C-PLFA fingerprint for methane oxidizing bacteria from a14 14insights. The C-PLFA fingerprints for the methane methane enriched agricultural soil (A). The C-PLFA fingerprintsfor a Type I and a Type II methanotrophic bacterium is shown forenriched agricultural soils showed that the mostcomparison (B) (M. album BG8 and M. trichosporium OB3b,active methanotrophic bacteria produced PLFAs thatrespectively).

eluted mainly in fraction 2, 4, 5, and 11 (Fig. 3A).

The radiolabelled fractions represent PLFAs with14

ECL values in the following intervals: 13.614.6, rophic PLFAs [8,20,22,23]. The C-PLFA finger-

15.316.1, and 17.717.9. Known fatty acids that print for a monoculture of a Type I methanotroph is

eluted in the same fractions were 14:0, 16:1, 16:0, shown in Fig. 3B (M. album BG8). PLFA and14

and 18:1. Approximately 77% of the radioactivity C-PLFA profiles for a Type II methanotroph are

associated with the soil PLFAs was recovered in depicted in Figs. 2 and 3B (M. trichosporium OB3b).

fraction 4 and 5 (e.g., 16:1 and 16:0). These fatty The above results suggest that the majority of the14acids are major constituents of Type I methanot- CH added to the methane enriched agricultural

4

Table 2

Concentrations of fatty acids indicative of methanotrophic bacteria in agricultural soil before and after methane enrichment

Signature Methanotrophs Before Methane After Methane

PLFAs Enrichment Enrichment21 21

nmol gdw nmol gdw

16:1v8c Type I ,0.01 5.9

18:1v8c Type II ,0.01 2.7


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soil was assimilated by organisms phenotypically imately twice as many degraders detected in the5 3

most similar to the Type I methanotrophic bacteria. latter environment (2.4 and 4.6310 degraders/ cm ,

Thus, Type I methanotrophs appear to have been respectively). These results were somewhat con-

responsible for more methane metabolism during the trasted by measurements of the capacity for aerobic14

incubation than the Type II methanotrophs. It is [ C]phenanthrene degradation in parallel samples14

noteworthy that this difference between bacterial (Table 3). Initial rates of [ C]phenanthrene degra-14

abundance and de facto population activity was not dation (measured as CO production) was 35 times2

revealed by the conventional PLFA analysis (Table greater in undiluted sludge samples than in marine

2). The discrepancy between population size and sediment samples (Table 3). After 24 h of incuba-

activity may be explained by cell specific differences tion, the rate of phenanthrene degradation increased

in kinetics and carbon conversion efficiency between significantly in the marine samples (data not shown).

Type I and II methanotrophic bacteria. Type I These results suggest that part of the phenanthrene

methanotrophs have been reported to be physiologi- degrader population in the marine sediment was

cally dominant in environments with an ample either in a dormant state or preferred substrates other14

supply of methane and nutrients as was likely the than [ C]phenanthrene during the initial 24 h of

case in the methane enriched agricultural soil incubation with the substrate.[22,24,25]. Bacterial phospholipid (PL) production relative to

poly-b-hydroxyalkanoate (PHA) production from a

common radiolabelled precursor may be used as a14

3.3. [ C]phenanthrene degradation simple indicator of metabolic status in microbial

communities [15]. This approach is based on the14

Degradation of [ C]phenanthrene in activated assumption that PHA synthesis occurs primarily

sludge and marine sediment was studied with differ- during unbalanced growth whereas PL synthesis isent radiolabelling techniques to obtain simultaneous an indicator of balanced growth mainly. Incubation

14information about the number of degraders, their of activated sludge with [ C]phenanthrene showed14

apparent physiological status, and their C-PLFA that assimilation of the label into PHA versus PL

fingerprints. Bacteria capable of degrading phenan- was affected by the oxygen status (Table 3). Rela-14threne were enumerated based on a three tube C- tively more label was assimilated into PHA under

14most-probable-number approach with [ C]phenan- oxic conditions compared to anoxic conditions

threne as the substrate (Table 3). This technique is (PHA/PL ratio of 0.67 and 0.3, respectively). These14

based on detection of CO produced from small findings correspond to PHA /PL ratios reported by2

14amounts of C-labelled substrates added to serially Dobbs and Findlay [15] for benthic marine com-

diluted samples e.g. [11,26]. The results show that munities before and after the introduction of physio-

aerobic phenanthrene degraders were relatively com- logical stress (PHA/ PL ratio of 0.3 and 0.6, respec-14

mon in both marine and sludge samples with approx- tively). These authors used [ C]acetate as a com-

Table 314

Characteristics of [ C]phenanthrene degradation in marine sediment and activated sludge

1 4 1 4 1 4 1 4Sample Incubation [ C]phenanthrene C-MPN C-PHA C-PL Lipid Ratio

aConditions Degradation

3 5 5 5 14 14 b(10 dpm/ml?h) (10 degraders / ml) (10 dpm / ml) (10 dpm / ml) C-PHA / C-PL

Marine Sediment Oxic 0.18 2.4 0.77 2.48 0.31

Activated Sludge Oxic 6.35 4.6 1.41 2.09 0.67

Activated Sludge Anoxic 0.06 N.D. 0.74 2.48 0.30

a 14 14Initial rate of [ C]phenanthrene degradation measured as CO production (018 h).

2bRelative amount of label assimilated into PLs to that assimilated into PHAs.

N.D.: Not Determined.


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mon lipid precursor to study whole community Approximately 6471% of the radioactivity associ-14

effects as a function of physical disturbances [15]. ated with C-PLFAs was recovered in fractions 414 14

The C-PHA/ C-PL ratio for aerobic phenanthrene and 5. These fractions represent PLFAs with ECL

degradation (0.67) suggests that the active bacteria values between 15.3 and 16.1 (e.g., 16:1 and 16:0)

may have been limited by one or several nutrients (Table 1).leading to unbalanced growth during the rapid Anaerobic phenanthrene degraders in activated

14substrate consumption under oxic conditions. The sludge produced a very different C-PLFA finger-

initial rate of phenanthrene turnover (18 h) was 105 print with 65% of the radioactivity recovered in

times greater for activated sludge incubated under fractions 2 and 15 (Fig. 5C). These fractions repre-

oxic conditions compared with samples incubated sent PLFAs with ECL values between 13.6 and 14.6,14

under anoxic conditions (measured as CO pro- and .19, respectively (Table 1).2

duction). Alternatively, two distinct groups of phen- The fingerprints obtained for aerobic and anaero-

anthrene degraders with different lipid composition bic phenanthrene degraders in activated sludge indi-

may have been involved in metabolism of the cate that two phenotypically very different bacterial

substrate under oxic and anoxic conditions. populations dominated the metabolism under oxic

It should be noted that the applicability of the and anoxic conditions (Fig. 5B and C). Thus,14 14

C-PHA / C-PL ratio as a strict measurer of meta- facultative anaerobic organisms may not have been

bolic status in environmental studies may depend on quantitatively important in the degradation of phen-

the choice of substrate in relation to the target anthrene in the sludge environment. In contrast, the

organism(s) as well as incubation conditions and fingerprints obtained with oxic sludge and oxic

incubation length. For example, interpretation of marine samples suggest that the physiologically most14 14

C-PHA / C-PL ratios may be obscured by the fact active aerobic phenanthrene degraders in these rather

that not all bacteria can produce PHAs e.g. [27]. In different environments were phenotypically related14

the present study, the simple C-lipid ratio appeared (Fig. 4A and B). The general PLFA composition that14

more useful as a supplement to the more detailed can be predicted from the C-PLFA analysis of

analysis of radiolabelled PLFAs. Interestingly, differ- these aerobic phenanthrene degraders contain com-14 14

ences in the C-PHA/ C-PL ratio for oxic and mon fatty acids found in a range of bacteria includ-anoxic incubations were paralleled by a shift in the ing pseudomonades and aeromonades [29]. Members14

C-PLFA fingerprint of the bacteria metabolizing of these genera have been shown to degrade14 14

[ C]phenanthrene (see below). The assimilation of [ C]phenanthrene in laboratory cultures [30].14 14

C-labelled substrates into C-PL alone can also be

3.5. Potentials and limitationsused as a measurer of microbial biomass production

during biodegradation [28]. This approach has been

Some microorganisms may not be detected withused recently to estimate total degradation (dissimila-14

the C-PLFA approach described here although theytion and assimilation) of the plasticizer di(2-1414 participate in the degradation of a given C-labelledethylhexyl)phthalate ([ C]DEHP) in soil and sludge

substrate. This will occur if the organisms catabolize[28].

the radiolabelled substrate but do not assimilate anyof the labelled intermediates. Such an uncoupling

143.4. Fingerprinting of [ C]phenanthrene can occur during e.g., co-metabolism of organic

degraders compounds. Co-metabolizing organisms may instead14

excrete labelled organic intermediates or CO that2

Marine sediment and activated sludge that de- may be assimilated subsequently by other organisms.14 14

graded [ C]phenanthrene aerobically had somewhat A non-specific assimilation of free CO produced2

different whole sample PLFA profiles (Fig. 4A and during the mineralization process may lead to14

B). In contrast, the C-PLFA fingerprints for another potential error which is labelling of organ-

aerobic phenanthrene degraders from the same set of isms that do not participate in the degradation of the14 14

samples were relatively similar (Fig. 5A and B). added C-substrate per se. An assimilation of CO2


10/13


1 4Fig. 4. Example of GC chromatograms of PLFAs extracted from environmental samples that degraded [ C]phenanthrene. A, marine

sediment incubated under oxic conditions; B, activated sludge incubated under oxic conditions. The fatty acids were analysed as methyl ester14

derivatives (PL-FAMEs) by capillary gas chromatography (extended chromatograms for simultanous detection of C-PLFAs). The14

corresponding C-PLFA fingerprints are shown in Fig. 5A and B.


11/13


environmental samples will, in most cases, dilute the14

CO substantially. As a result, autotrophic fixation2

will appear negligible relative to the direct hetero-14

trophic assimilation of C-labelled organic metabo-

lites. Addition of surplus unlabelled CO prior to the2onset of the experiments may further reduce this

error. The potential errors associated with non-spe-

cific labelling will depend on the substrates used and

should be evaluated with proper controls. However,

it is worth noticing that metabolic interactions among

organisms may be a prerequisite for complete miner-

alization of many organic compounds. Such interac-14

tions may be revealed by analysis of C-PLFAs.

Experiments performed in our laboratory with miner-14 14

alization of [ C]methane, [ C]phenanthrene and14

[ C]DEHP suggest that one group of organism oftendominate the metabolism of a given substrate and

that the obtained fingerprints are most often specific

and highly reproducible among samples.14

Refinements of the described C-PLFA method

may include: (i) shorter incubation time in the

presence of the labelled substrate to avoid potential

enrichment effects, (ii) greater resolution of labelled

PLFAs, (iii) automatic detection of radiolabelled

PLFAs. The latter may be accomplished by use of

e.g., automatic solid or flow scintillation detection in

combination with either GC or HPLC analysis e.g.,[18,32]. Such detection systems may also allow

14quantification of individual C-PLFAs and thereby

increase the resolution of the fingerprints. Alter-13 14

natively, use of C-labelled instead of C-labelled

substrates may allow GCMS detection of individual13

PLFAs enriched in C [31,33].

Despite the above limitations, variations of the14

present C-PLFA fingerprinting method appear use-

ful as a tool in environmental studies of microbial

turnover of a specific carbon substrate. Similar to

other studies based on isotopic analysis of cell lipidbiomarkers e.g. [19,31,33,34], the present method

1 4 14 will be especially helpful for studies of substrateFig. 5. C-PLFA fingerprints for [ C]phenanthrene degradingmetabolism in complex microbial communities. Onemicroorganisms from marine sediment incubated under oxic

conditions (A), activated sludge incubated under oxic conditions attractive feature of these strategies is that they do(B), and activated sludge incubated under anoxic denitrifying not require prior enrichment or isolation in order toconditions (C).

get phenotypic information about a specific group of14

organisms. The present C-PLFA fingerprinting

occurs in autotrophic organisms and via anaplerotic method is based on consumption and assimilation of

reactions in heterotrophic organisms [31]. Fortunate- radiolabelled carbon by the physiologically most2ly, the background of unlabelled CO and HCO in active microorganism involved in the metabolism of

2 3


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14 [2] E.L. Madsen, A critical analysis of methods for determininga given C-labelled substrate. Although dormant orthe composition and biogeochemical activities of soil micro-resting bacteria may also metabolize small amountsbial communities, in: G. Stotzky, J.-M. Bollag (Eds.), Soil

of exogenous substrate [35,36], the current method Biochemistry, Marcel Dekker, New York, 1996, pp. 287will predominantly detect vegetative (actively grow- 370.

[3] C.J. Hurst, G.R. Knudsen, M.J. McInerney, L.D. Stetzen-ing) bacteria due to the much greater cell specificbach, M.V. Walter, Manual of environmental microbiology.metabolic rates relative to the rates in non-growingAmerican Society for Microbiology, Washington, 1997.

cells. Potential method applications include: (i)[4] P. Vandamme, B. Pot, M. Gillis, P. De Vos, K. Kersters, J.

comparison of active microbial degrader populations Swings, Polyphasic taxonomy-a consensus approach to bac-terial systematics, Microbiol. Rev. 60 (1996) 407438.in different environments, (ii) studies of changes in

[5] M. Goodfellow, A.G. ODonnell, Chemosystematics: cur-degrader population activity following changes inrent state and future prospects, in: M. Goodfellow, A.G.

environmental conditions, (iii) monitoring the selec-ODonnell (Eds.), Chemical methods in prokaryotic

tive enrichment of microorganisms that are physio- systematics John Wiley and Sons Ltd., Chichester, 1994.logically dominant under in situ conditions. In the [6] D.C. White, Is there anything else you need to understand

14 about the microbiota that cannot be derived from analysis oflatter case, the C-PLFA fingerprinting methodnucleic acids?, Microbiol. Ecol. 28 (1994) 163166.would make it possible to evaluate whether a new

[7] R.H. Findlay, F.C. Dobbs, Quantitative description of micro-

bacterial isolate did indeed play any significant role bial communities using lipid analysis, in: P.F. Kemp, B.F.in the turnover of a given substrate in the original Sherr, E.B. Sherr, J.J. Cole (Eds.), Handbook of Methods in

14sample. Phenotypic information based on the C- Aquatic Microbial Ecology, Lewis Publishers, Boca Raton,

Florida, 1993, pp. 271284.PLFA fingerprints may also aid in designing enrich-[8] J.B. Guckert, D.B. Ringelberg, D.C.White, R.S. Hanson, B.J.ment conditions that favour growth of selected

Bratina, Membrane fatty acids as phenotypic markers in themicroorganisms.

polyphasic taxonomy of methylotrophs within the proteobac-In summary, the present method based on specific teria, J. Gen. Microbiol. 137 (1991) 26312641.

14radiolabelling and C-PLFA analysis appears prom- [9] A. Tunlid, D.C. White, Biochemical analysis of biomass,

community structure, nutritional status, and metabolic activi-ising as a relatively simple tool for studying micro-ty of microbial communities in soil, in: G. Stotzky, J.-M.organisms in mixed microbial communities. BecauseBollag (Eds.), Soil Biochemistry, Marcel Dekker Inc., New

of the detection of physiologically dominant micro-York, 1992, pp. 229262.

14

organisms mainly, the C-PLFA fingerprinting ap- [10] I. Sundh, P. Borga, M. Nilsson, B.H. Svensson, Estimation ofproach may complement other methods used for cell numbers of methanotrophic bacteria in boreal peatlands

based on analysis of specific phospholipid fatty acids, FEMSdirect detection and characterization of microorga-Microbiol. Ecol. 18 (1995) 103112.nisms in complex environments.

[11] C.C. Sommerville, C.A. Monti, J.C. Spain, Modification of

the 14C most-probable-number method for use with nonpolar

and volatile substrates, Appl. Environ. Microbiol. 49 (1985)

711713.Acknowledgements[12] S.O. Petersen, M.J. Klug, Effect of sieving, storage, and

incubation temperature on the phospholipid fatty acid profileWe thank Kirsten Maagaard for her excellent of a soil microbial community, Appl. Environ. Microbiol. 60

technical assistance and Sren O. Petersen for help- (1994) 24212430.[13] R.H. Findlay, D.C. White, A simplified method for bacterialful suggestions. This work was supported by the

nutritional status based on the simultaneous determination ofEuropean Commission grant BIO4-CT96-0419 and phospholipid and endogenous storage lipid poly-b-hydroxy-the Danish Technical Research Council grant

alkanoates, J. Microbiol. Methods 6 (1987) 113120.9502651. [14] J. Fang, R.H. Findlay, The use of a classical lipid extraction

method for simultanous recovery of organic pollutants and

microbial lipids from sediment, J. Microbiol. Methods 27

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