FEMS Microbiology Ecology 26 (1998) 17-26

Diversity in methane enrichments from agricultural soil revealed by DGGE separation of PCR amplified 16s rDNA fragments

Sigmund Jensen ‘, Lise 0vreb ‘3 *, Frida Lise Daae b, Vigdis Torsvik ”

Received 17 October 1997: revised 2 February 1998: accepted 5 February 1998

Abstract

Denaturing gradient gel electrophoresis (DGGE) profiles of PCR amplified V3 regions of 16s rRNA genes were used to assess the diversity in enrichment cultures with methane as the only carbon and energy source. The enrichments originated from two agricultural soils. One was a sandy soil with low (10%) organic content, the other an organic soil with approximately 50% organic content. DGGE provided a fast evaluation of the distribution of amplifiable sequence types indicating that specific bacterial populations had been enriched from each soil. The DGGE profiles revealed a broader range of amplified V3 fragments in the community derived from organic soil than from sandy soil. Fragments from 19 individual DGGE bands were sequenced and compared with 27 previously published 16s rRNA gene sequences. The sequences confirmed the high diversity with the presence of different methylotrophic populations in each enrichment. No affiliation was found with type I methanotrophs, instead type I1 methanotroph sequences were found in the enrichments from both soil types. Some of the fragments from the organic soil enrichment were not affiliated with methylotrophs. Most of the sequences clustered distantly on a branch within the a-Proteobacteria. These facts suggested that previously undescribed methylotrophs are abundant in methane enrichments from agricultural soil. 0 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.

ki~~~~r/r: Soil: Enrichment; Mcthanotroph; Denaturing gradient gel electrophoresis: Phylogeny

1. Introduction

Methane oxidation in soil environments, which is carried out by key organisms, is estimated to account for 80% of the methane consumed globally [l]. The organisms both limit methane emissions from anae- robic zones to the atmosphere and consume atmos- pheric methane [2,3]. The main sinks for methane in

soils are believed to be aerobic methanotrophic bac- teria [4,5]. Methanotrophs can be cultivated on methane as the sole carbon and energy source in mixtures with air containing lo-50% methane [6]. but it is unclear to what extent high methane con- centrations provide growth of methanotrophs capa- ble of low methane consumption [7.8].

Liquid methane enrichments are useful for charac- terization of methanotrophs because these organisms

* Corresponding author. Fax: +47 55 589671; do not always grow very well on solid media [9%11]. E-mail: lise.ovreas@im.uib.no In addition mixed cultures offer the possibility of

0168-6496198 1$19.00 (Q 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PIISO168-(,496(9X)00017-8

syntrophic relationships and hence growth of other- wise nonculturable bacteria may occur [13]. Charac- terization of isolates obtained from enrichments of methanotrophs from soil and water samples has led to the establishment of five groups of methano- trophic bacteria [13], recently revised by Bowman et al. [14]. Conclusive identification, however, was difficult due to the restricted use of substrates by the methanotrophs. Phylogenetic studies now allow such metabolic characteristics to be circumvented since it has been found that methylotrophic bacteria can be classified according to 16s rRNA gene homology [15]. A phylogenetic tree based on se- quence similarities revealed that the methylotrophs were separated into three coherent clusters of the Proteobacteria. c(, B and y, in which bacteria in each cluster shared physiological characteristics.

Molecular analysis of communities and enrich- ments offers a great potential for the study of un- isolated bacteria that can be enriched under specific conditions. PCR amplification of 16s rDNA with subsequent denaturing gradient gel electrophoresis (DGGE) offers resolution of communities that are PCR amplifiable [16-211. In agricultural soils meth- anotrophic bacteria are most likely not a dominating part of the microbial community. Therefore enrich- ments under specific conditions are required prior to PCR-DGGE analyses of these bacteria. The DGGE profile visualizes all amplified gene variants and sim- plifies the task of detecting specific 16s rRNA gene sequences (i.e. specific bacteria [Xl). DGGE screen- ing has the potential to reveal how efficient methane enrichments are for retrieval of methane consumers from the environment. This strategy will be of im- portance for those organisms that have to be en- riched under very specific conditions.

The DGGE method is particularly useful as an initial investigation for distinguishing between com- munities and identifying the numerically dominating community members. The method enables us to rap- idly screen multiple samples and obtain valuable in- formation about community changes and differences. Furthermore, it can be used to follow enrichment cultures and to control isolation methods in order to evaluate the efficiency of these methods for cap- turing the numerically dominating members of com- munities.

The work presented in this paper is part of an investigation on changes in methane oxidation ca- pacity and populations of methanotrophic bacteria in response to changes in land use and agricultural management. Our goal was to characterize the bac- terial diversity in methane enrichments as revealed by DGGE profiles of 16s rDNA from the numeri- cally dominating populations. Further we wanted to use this approach for comparing populations of methanotrophic bacteria from different soils sub- jected to different agricultural managements.

2. Materials and methods

The samples used as inocula for the methano- trophic enrichment cultures studied were taken in January 1995 from two agricultural fields at Stend, south of Bergen, Norway. The sandy soil field, Sto- rakeren (Sto), has been cultivated for approximately 125 years. It has been used for intensive agricultural production, frequently tilled and fertilized with about 50 000 kg of manure and about 150 kg of mineral N (NH:, NO,) haa’ for the last 10 years. The other field, Krohnestykke (Kro), was a bog until the beginning of this century, when it was brought into agricultural use. For the last 10 years it has been used as a pasture and the manuring and mineral fertilization has been less intensive than for Sto- rakeren. Both soils were covered with a thin layer of snow at the time of sampling. Samples were col- lected beneath the plant cover, from 5 cm depth in Krohnestykke and from IO cm depth in Storikeren because of frost in the uppermost soil layer. Five subsamples were obtained along an approximately 50-m transect in each field, and the samples were pooled and mixed. The soils were characterized as described earlier [23], with total cell numbers ob- tained as in Porter and Feig [24]. For both soils a low consumption of atmospheric methane was ob- served as suspected. Chemical and biological charac- teristics are given in Table 1. The soil samples were stored in black polyethylene bags at 4°C for approx- imately 6 weeks before the enrichment cultures were initiated.

2.2. Methane enrichment cultures

Modified NMS medium (10 ml, pH 6.8) in 120-ml serum bottles capped with butyl rubber stoppers was inoculated with 5 g of soil [13]. The medium was modified with trace elements recommended for Methylomonas rnethanicn by ATCC ( 1306), EDTA ferric monosodium salt, and different concentrations of CaClZ*2H20 (0.134 g 1-l). KHzPOd (0.272 g 1-l) and Na2HPOJ*2HZ0 (0.356 g 1-l). All cultures were supplied with methane; high methane enrichments by withdrawal of 20 ml air and injection of 22 ml CHJCOZ (90: 10) mixture and low methane enrich- ments by direct injection of 20 ~1 CHd and 2 ml CO?. The cultures were incubated in the dark in shaking water baths (85-90 rpm) at 15°C or 30°C. Subse- quent subculturing was done when the cultures be- came visibly turbid (every l-2 weeks), by transfer- ring 1 ml culture to 4 ml fresh medium in similar bottles with the appropriate headspace gas mixtures. Cell morphology was examined after 12 weeks (eight subculturings) by microscopy of acridine orange (AO) stained cells [25]. The cell viability was exam- ined by growth on NMS plates (2% Bacto agar). incubated for 2 weeks at 30°C in an atmosphere enriched with CHd/COZ (90: 10).

After 31 weeks (20 subculturings) the enrichment cultures were harvested by centrifugation of 1..5-ml samples at 10 000 X g for 30 min. The cell pellets were washed once in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na?HPO,.7H?O, 1.4 mM KH?PO.k [pH 7.3]), resuspended in 50 ~1 sterile distilled water, and frozen. The cell suspensions were thawed and used for whole cell PCR amplification.

2.3. PCR arnpl$ication

A highly variable region (V3) of the 16s rRNA gene [26] was amplified using PCR primers. One pri- mer complemented a region conserved among mem- bers of the domain Bacteria (Escherichiu coli posi- tions 338-358; 5’-CGCCCGCCGCGCGCGGCG- GGCGGGGCGGGGGCACGGG GGGACTCC- TACGGGAGGCAGCAG-3’) [2 l] and incorporates a 40-bp GC clamp. The specificity of this primer is imparted by the underlined region. The other primer is based on a universally conserved region (E. co/i positions 517-534; 5’-ATTACCGCGGCTGCTGG-

3’) [17]. This primer combination amplified a 233-bp fragment suitable for subsequent DGGE analysis. 10 pl of washed cell suspension was added directly to 0.5 pM of each primer, 100 FM of each deoxynu- cleotide triphosphate. 2% bovine serum albumin (BSA), 10 ~1 lOXRed Hot buffer, 0.5 units of Red Hot DNA polymerase (Advanced Biotechnologies Ltd., UK), and sterile water to a final volume of 100 ~1. PCR amplification was performed in an au- tomated thermal cycler (9600 Perkin ElmerlCetus) with an initial denaturation at 92°C for 2 min fol- lowed by 30 cycles of 1 min denaturation at 92”C, 30 s annealing at 55°C and 2 min extension at 72”C, before the final extension at 72°C for 6 min.

.?. 4. DGGE analysis

DGGE was performed with a Hoefer SE600 ver- tical dual cooler system. Polyacrylamide gels were made in a denaturant gradient maker with 8% (w/ v) polyacrylamide gels (bisacrylamide gel stock sol- ution. 37:5: 1; Bio-Rad Laboratories Inc.), giving a denaturing gradient ranging from 15 to 45% (100% denaturant corresponds to 7 M urea and 40%) (v/v) formamide deionized with AGSOl-X 8 mixed-bed resin (Bio-Rad)). Gels were allowed to polymerize overnight. Electrophoresis was run in 0.5 XTAE buffer (20 mM Tris, 10 mM Na-acetate, 0.5 mM NazEDTA [pH 7.41) initiated at 20 V for 10 min before a 3-h continuous run at 200 V and 60°C. The gels were then stained for 45 min in TBE buffer (89 mM Tris-borate [pH 8.01, 89 mM boric acid. 2 mM NaZEDTA) containing 1: 10 000 concentrated SYBR Green1 (Molecular Probes, Eugene, OR, USA) and rinsed in a water bath for 5 min before photographed on a UV transillumination table with a Polaroid MP4 Land Camera (Polaroid Corpora- tion, Cambridge, MA, USA).

2.5. Sequencing of‘ DGGE buds

Small pieces of selected DGGE bands were punched from the gel using sterile pipette tips. The pieces of acrylamide containing the bands were placed in sterilized vials and 20 ~1 sterilized water was added. The samples were subjected to passive diffusion overnight at 4°C. Tenfold dilutions of the eluate was then used as template DNA in a ream-

plification PCR using the same primers and reaction conditions as before. The highest dilutions that cre- ated ample PCR products were rerun on the DGGE to confirm their positions relative to the bands from which they were excised. These reamplifications con- sisted of only one band with no background of other bands observed. The PCR products were then di- rectly sequenced using the AmpliCycle sequencing kit according to the manufacturer’s directions (Per- kin Elmer, Roche Molecular Systems. New Jersey, USA). The products from the sequencing reactions with incorporated radioactivity were then separated on 6% (w/v) acrylamide-bisacrylamide ( 19 :7 : 1) and 7 M urea wedge-shaped gradient gels (0.2-0.6 mm) in the LKB 9010 Macrophore sequencing system. The gels were run at 55°C for 3 h at 1750 V. The gels were washed for 70 min in 10% acetic acid. ?O min in 10% acetic acid and 1% glycerol, and then quickly rinsed in distilled water and dried over- night on a glass plate. Detection of sequences was performed by exposing the gel to Hyperfilm-P max (Amersham) overnight.

The 16s rRNA sequences from the enrichment cultures were aligned with parts from complete 16s rRNA sequences of methylotrophic bacteria ob- tained from the EMBL gene bank. Sequence align- ments were performed using the PILEUP program from the University of Wisconsin Genetics Com- puter Group (GCG programs, version 8.1). Matrices of evolutionary distances were computed using the program Dnadist from Phylip version 3.5 [37] with the Jukes-Cantor model, which assumes independent change at all sites with equal probability [28]. Phy- logenetic trees were constructed from the evolution- ary distances using the Fitch-Margoliash algorithm [29]. The phylogenetic trees were checked by boot-

T‘ablc I Characteristics or the soil inoculum

Site CH, Texture

oxidation,’

Km 1.74+0.71 organic

strap analysis (100 data sets) using the program SEQBOOT. Bootstrap values indicate the frequency of resampling that supports a specific branching pat- tern.

3. Results

Initially enrichments were set up using two con- centrations of methane and they were incubated at two different temperatures. Incubation at 30°C re- sulted in lower cell densities with low than with high methane concentration. A0 cell counts showed that 1 .O X lOi cells ml-’ were present in the organic soil enrichments and 8.3 x 10’ cells ml-’ in the sandy soil enrichment. This was less than 10% of the cell numbers found in enrichments with high methane concentration (3.4X 10’ cells ml-’ in the organic soil and 1.3X IO!’ cells ml-’ in sandy soil). Incuba- tion at 15°C also resulted in low cell densities, but with no significant difference between high and low methane concentrations. A total of six cell morpho- logical variants were observed in the sandy soil en- richments with an additional two variants found in the organic soil enrichments. In the high methane enrichments at 3O”C, detectable methanotrophic ac- tivity was observed after 7 days of incubation as decreased headspace concentrations of methane. These enrichment cultures were chosen for furthel investigations.

Gas chromatographic analysis showed that both enrichments consumed about 95% of the added methane within a week. Methane was required for growth. and the cultures usually reached an OD at 540 nm of about 1 and a viable cell number of lO”- IO’ ml-’ indicating the presence of methanotrophs.

2.06 6.4 I64 4.7

sto 1.5SfO.l9 sandy loam 9.1 5.38 0.46 5.6 36 7.9

5

6-

7-

R_

IO-

A B 3.2. DGGE anal~xis oj unlpl$ed rDNA from the

Fig. I. DGGE separation pattern of PCR amplified 16s rDNA fragments (position 338-517) from enrichment cultures from two different soil samples. A: Organic soil (Kro). B: Sandy soil (Sto). Labelling patterns are for bands that were excised, reamplified

and sequenced. The denaturant gradient is 1545%.

The colonies growing on NMS Bacto agar plates were < 1 mm in diameter and transparent, or l-5 mm in diameter and colored. Bacteria from both enrichments grew as white and pink colonies, and in addition yellow colonies were observed from the sandy soil enrichment. Microscopic examination showed that the enrichment from sandy soil con- tained two dominant morphological types (rods and cocci), whereas that from the organic soil con- tained four additional dominant variants (smaller rods, cells with stalks. chains of rod-shaped cells, and big polar flagellated cells, probably eukaryotes). After continued re-streaking on plates only one col- ony type for each enrichment was observed. The col- onies from sandy soil enrichment were pink. contain- ing curved rod-shaped non-motile Gram-negative bacteria, whereas those from the organic soil enrich- ment were ‘off-white’ with non-motile Gram-negative cocci.

Two parallel enrichments were used for DGGE screening. They both produced identical DGGE pat- terns. Only one of the parallels was used for further analysis with reamplification and sequencing. Sepa- ration of DNA fragments by DGGE revealed band- ing patterns characteristic for each enrichment cul- ture (Fig. 1). In the enrichment from organic soil, 14 bands dominated. These were spread from a dense cluster at about 27% denaturant down into well sep- arated bands as far as 43% denaturant. The enrich- ment from the sandy soil had 13 dominant bands more uniformly separated in a narrower range of the denaturant gradient gel. Four bands from the two enrichments (Kro3 and Stol2, and Kro4 and Stol5) showed up at the same position. meaning that they consisted of fragments with similar melting behavior. The rest of the bands’ positions were unique to each enrichment. The varying brightness of the bands reflects their varying content of DNA fragments.

3.3. hterpretation of phylogenetic relationships

To investigate which organisms were growing in the soil enrichment cultures, fragments from a total of 19 selected DGGE bands were sequenced and subjected to phylogenetic analysis. Fig. 2 shows the phylogenetic relationship of distinct sequences in combination with 16s rRNA reference sequences of methylotrophic bacteria retrieved from the EMBL gene bank. The majority (12) of the sequences clus- tered within a-Proteobacteria. Seven of the nine Sto sequences clustered within this group, and their phy- logenetic positions are indicated by the Sto17 and Sto19 sequences. They showed closest affiliation to type II methanotrophs. The Kro sequences that clus- tered within the a-Proteobacteria group (5 out of 10) had their closest affiliation to Meth~~lohucteriu,~z sp. (indicated by Kro5 and Kro9). Sequence similarities within the enrichments were higher than between the enrichments. Only two sequences were found closely affiliated with the reference sequences ( > 90% simi- larity). These were Sto19, which showed homology to Methylosinus sp., and Sto13. which showed ho- mology to Meth~~lohacillus sp. The phylogenetic po-

72

94 1

- Rhodopseudomonas palusiris

- Bradyfiizobium sp.

Pseudomonas festosteroni

Mefhylosinus sp. LAC 0

Mefhylocysbs paws OBBP l

Mefhylosinus lrichospotium OB3b 0

Methylosinus sporium 0

Methylobacterium extorquens 0

Mefhylobacterium sp. PR-6 0

Methylobacfetium sp. PK.1 0

KroS

Mefhylobacferium oganophilum XX 0

I- !jto13 - Mefhylobacillus flagellafus KT-1 n

I Mefhylomonas mefhanolica u

95 I Mefhylobacillus glycogenes J

Methylophilus methylotmphus AS1 q

Mefhylomonas mefhylovore q

d- Agrobacterium fumefaoens

Rhodopseudomonas acidophila

,- Rhodospirikim rubrum

-I I Kroi

0.1

a

I

p

I

/a

v--j- Mefhylomonas methanica I BY

Mefhylomonas sp. A4 I

Mefhylomonas rubra I

S. Jensen et ul. I FEMS Mcrohiology Ecology 26 (1998) 17-26 23

Fig. 2. Unrooted phylogenetic tree based on fragments of 16s rRNA gene sequences showing the phylogenetic relationship of methylotro- phic bacteria. Reference sequences used in preparing this figure were derived from the EMBL gene bank. Bootstrap values are indicated

(see text for explanation). The scale bar represents 0.1 estimated nucleotide changes per sequence position. Three different subdivisions of the Proteobacteria, i.e.. CI, p and y, are indicated in the figure. The symbols refer to physiological characteristics of the methylotrophs ac- cording to the pathway used for formaldehyde assimilation: serine pathway utilizing methanotrophs (0) and non-methane oxidizing meth- ylotrophs (0): ribulose monophosphate pathway utilizmg methanotrophs (m) and non-methane oxidizing methylotrophs (0). t

sitions of sequences not affiliated with the reference organisms are indicated by Krol . The four sequences found near this position were all from the organic soil enrichment (Krol4).

4. Discussion

In the present work, DGGE of PCR amplified 16s rRNA fragments was exploited for analyzing the community structure and diversity in enrichment cul- tures of methanotrophic bacteria. These bacteria are very difficult to isolate but can be readily enriched. Molecular biological methods therefore have brought a breakthrough in community analysis of methanotrophic bacteria, applying DNA extracted either directly from environmental samples or from enrichment cultures [30-321. However, until now these investigations have been based on probing and sequencing of cloned 16s rRNA gene libraries, a strategy that is very laborious and time consuming.

Earlier investigations of methylotrophic phylogeny have included the complete 16s rRNA molecule for strain identification. We therefore tested the possibil- ity of using only the V3 region to obtain a valid methylotrophic phylogeny. Sequences from 20 meth- ylotrophic reference organisms previously sequenced by Bratina et al. [33] and seven non-methylotrophic organjsms were retrieved from the gene bank and approximately 300 bp aligned to enable construction of distance matrices. The phylogenetic tree obtained (Fig. 2) was in agreement with the main findings of Bratina et al. [33], and the positions of the reference organisms were in agreement with Tsuji et al. [15] and Brusseau et al. [34]. Therefore we regard the V3 region as suitable to readily assess the phyloge- netic relationships between these reference organisms and unknown methylotrophs.

We were careful to process the two methane en- richments as equally as possible to enable observa- tions of differences originating from the inoculum.

The enrichment conditions applied were highly selec- tive for physiological types that could sustain growth on methane as the sole carbon and energy source [35]. However, cocultivation of non-methylotrophs seems difficult to exclude. Two parallel enrichments were analyzed by PCR-DGGE. CO? was added to support methanotrophs having the serine cycle and methanotrophs equipped with the Calvin cycle [6]. Therefore. in theory, autotrophic oxidizers of NH:, CO, Hz, HzS, or phototrophs [36] could have been sustained to some extent. However, the methane ox- idation activity and the high cell densities indicated a high presence of methanotrophs. Pigmented colonies that grew to a size of 5 mm in an atmosphere sup- plemented with methane accounted for lo”-lox cfu ml-’ and were assumed to consist of methanotrophs. In transmission electron microscopy of cells from both enrichments, intracytoplasmic membranes were seen arranged around the periphery of cells, indicating the presence of type II methanotrophs. Examination of cell shapes found in the enrichment cultures indicated that the organic soil (Kro) sus- tained the highest diversity of organisms. This is in agreement with earlier observations of heterotrophic diversity in these soils [37].

The V3 regions of bacteria from the enrichment cultures were PCR amplified with primers previously determined to be specific for all Bacteria. Previous findings revealed these primers to detect template variants constituting down to 1% of a population [l7]. Differently positioned bands consist of DNA with different melting domains and different sequen- ces, hence judged to be derived from different species [17,20,38]. However, due to 16s rDNA heterogene- ity, different bacteria often generate more than one band. Since an aliquot from the enrichment cultures was amplified directly, this should ensure capturing the most dominating templates present. However, problems could have occurred due to PCR bias to- wards some template variants, giving a high yield of these and low yields of others [39.40]. Also produc-

tion of multiple fragments due to co-amplification of 16s rDNA molecules from different bacteria could have occurred. The primers, however, were not de- generated and unlikely to give extra fragments [41]. As only minor amounts of chimeric fragments were likely to be present after PCR [43]. it is probable that the fragments amplified reflect the major proportion of fragments present in the enrichment cultures.

In total more than 20 different bands were seen on the DGGE giving a distinct pattern for each enrich- ment culture. The bands from the organic soil en- richment were widely spread along the gradient, whereas the sandy soil enrichment gave several in- tense bands within a narrower gradient range. This suggested the highest diversity among fragments from the organic soil and fewer dominating fragment variants in the sandy soil. The DGGE patterns are a display of the numerically dominant populations. The pattern therefore provides information on spe- cies richness only when the populations are equally abundant. A few numerically dominant populations may easily disguise a greater species richness. Some bands had the same relative position on the DGGE, and could therefore be derived from the same organ- isms. By sequencing analysis we found that this was not true. Kro4 and Sto 15 were located at the same position, but were different. Kro4 had a very low phylogenetic alhliation to any of the sequences in the database and clustered together with Krol. while Kro5 clustered with Kro17 in a branch distantly af- filiated to the type 11 methanotrophs. Apart from Krol4, all bands were affiliated with the methylo- trophs. Sto19 showed high similarity (97%) to the genus Mrt/z~h~sirzu.s and Sto 13 high similarity (94%) to Metlz~~lohu~i1lzr.s. The presence of hfrth~hirm sp. in both cultures was supported by the observed cell and colony morphology (rods in white to yellow col- onies), and electron microscopy observations. Type II methanotrophs seem common in soil [43] and soil enrichments [34], but no close homology to type I methanotrophs was observed in this study. Remark- able, however, was the discovery that most of the sequences clustered distantly on a branch within the a-Proteobacteria. This was found for seven of the nine sandy soil enrichment sequences and five of the IO organic soil sequences. The closest relatives were type II methanotrophs and ~l~th~~loh~lc~tr~iz~t?l

sp. If any bacteria represented by these sequences

can grow on methane, then they represent novel methanotrophs.

The present work shows that methanotrophic bac- teria are difficult to cultivate and isolate. Even though several types of bacteria form colonies upon initial plating from the enrichment cultures, only one type from each enrichment was successfully retained after transfer to new plates. As very little information on the diversity and community struc- ture of methylotrophs could be gained from isolates. this was not explored further. Instead we focused on analyses of methylotrophs in enrichment cultures us- ing the culture independent PCR-DGGE method combined with sequencing. This method proved to be much more labor efficient and provided more in- formation than the culturing technique. This is also a much easier and more rapid approach for analyzing methanotrophs in environmental samples than se- quencing cloned PCR products which has been used until now [30.31].

To our knowledge this is the first study where DGGE analyses of PCR amplified 16s rRNA gene fragments have been used to study diversity and community structure of methylotrophic bacteria. The method has great potential in monitoring differ- ences and changes in the diversity of methylotrophic bacteria in soil and other habitats. The method pro- duced a distinct DGGE profile for each methane enrichment culture and revealed the organism diver- sity in the cultures to be higher than found from morphological observations.

Acknowledgments

We thank Andrew Holmes for advice during con- trol of secondary sequence structure and Colin Mur- rell for constructive comments on the manuscript. This work was supported by funds provided by the Norwegian Research Council to S.J. and L.0.

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