Aerobic methanotrophic bacterial communities in

Aerobic methanotrophic bacterial communities in sediments of Lake Constance

Dissertation

zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) an der Universität Konstanz, Fachbereich Biologie

vorgelegt von

Monali Rahalkar Konstanz 2006

Tag der mündlichen Prüfung: 21. Februar 2007

Referent: Prof. Dr. Bernhard Schink Referent: Prof. Dr. Andreas Brune Konstanzer Online-Publikations-System (KOPS) URL: http://www.ub.uni-konstanz.de/kops/volltexte/2008/2378/ URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-23787

Dedicated To My Dear Parents Who have really done a lot for me….

Acknowlegements This work was carried out under the supervision of Prof. Bernhard Schink in the laboratory of Microbial Ecology and Limnology chaired by him in the Faculty of Biology, University of Konstanz, from April 2003 to December 2006. This work is a part of the Sonderforschungsbereich (SFB) 454 program-B1 subproject, supported by the Deutsche Forschungsgemeinschaft. Words of thanks are less to express my gratitude towards Prof. Schink for giving me an opportunity to work with him as his PhD student. He is a great ‘Guru’!! (Teacher: in sanskrit). His continuous enthusiasm about my work, support and deep knowledge in various aspects of Microbiology has been a driving force for me. I will always remember the scientific discussions with him and his suggestions throughout my life. Next, I would like to thank Ingeborg Bussmann for helping me to learn different techniques in the cultivation of methanotrophs, especially gradient cultivation. Her contribution to the methane and oxygen measurements and fruitful discussions is gratefully acknowledged. I take the opportunity to thank Dr. Michael Hoppert, University of Göttingen, for performing electron microscopy of our strain. I am grateful to Alfred Sulger and his colleagues from the Limnology section for their help in sample collections. I would further like to thank Prof. Peter Kroth for allowing me to use the Real time PCR machine and also for his help from time to time. I would also express my sincere thanks to Prof. Adamska for allowing me to use glass desiccators, which were really precious for growing my bacteria. Also the help from the members of her group is gratefully acknowledged.

I would like to thank Prof. Andreas Brune for being a referee for my thesis, in spite of his busy schedule. I would also thank him for his advices and suggestions when he was there in Konstanz. I would like to thank Uli Stingl, Dirk Schmitt-Wagner and Oliver Geissinger for their help when I was learning to use ARB. Thanks to Claudia Wilderer for her help during agar shakes. I also would like to thank Katrin Styp von Rekowskii for her help when I checked if my methanotroph was producing any AHLs. I also thank Christian Bruckner for his help during the nitrogenase assay. Thank you Paula for your friendly advice, support and laugh. Sascha, thanks for the friendly conversations and your sense of humor. Further I would also like to thank Jörg Deutzmann for sharing his ideas, discussions and for translating the summary in german. Thanks to Ansgar Gubner from A G Kroth for the help in translation of some difficult sentences. I thank Britta for her last minute tips during the thesis preparation. I also thank Simone Gerhardt for her encouragement and wishes. I express my sincere thanks to all my present and previous lab members and Bodo Philipp, from our group for creating a nice environment and for timely help. Thanks to all my Indian friends especially Shanmu and Geetika for their help and of course to Farzana, for being always there with a helping hand. Here I want to express my love and gratitude to my parents, my brother, inlaws and other family members and friends. Their constant support, wishes and care have been very important for me. |Last but not the least; it’s my dear husband, Rahul. Without his care, support and help in everything, this thesis was just not possible.

Abbreviations pMMO – particulate methane mono-oxygenase sMMO – soluble methane mono-oxygenase pmoA – gene for alpha subunit of the pMMO 16S rDNA – gene coding for small subunit of ribosomal ribonucleic acid MOB – methane oxidizing bacteria EPS – extracellular/exopolymeric substance LO – cells grown under low oxygen tension HO – cells grown under high oxygen tension MO – cells grown under moderate oxygen tension ICM – intracytoplasmic membrane MS – membrane stacks ANME – anaerobic methane oxidizing archaea SRB – sulfate reducing bacteria

Preface Bacteria are among the most fascinating organisms in the world. The word ‘incredible’ is appropriate for them, because they can do those processes which modern machines or reactors cannot do. With the advent of sequencing their whole genomes many exciting facts have been revealed. Several metabolic pathways, suite of enzymes are well packaged and controlled within the genomes within a small cell of about one µm diameter. This quality makes them the key players in element transformations, e.g. C, N, P, etc. Bacteria are the silent actors, transforming different forms of elements back and forth and cycling them, making life on Earth feasible. Microbiology started with cultivation of bacteria in the laboratory and by studying their biochemical properties, so as to predict what metabolic processes they carried out in nature. Now with the help of molecular biological tools like cloning, analyzing metagenomes, many things can be studied or predicted without cultivation. Cultivation remains still the method of choice because it opens the doors to infinite amount of information lying within the bacterium. With the notion that only 1% or less of the total bacteria can be cultivated the remaining 99% uncultured bacteria remain a challenge for microbiologists. It can be said that amidst all the difficulties in cultivation, these uncultivated groups are like a ‘mirage’ which microbiologists like to follow. My studies on methanotrophic or methane oxidizing bacteria (MOB) thus focus on improving the cultivation strategies of ecologically relevant methanotrophs by providing them more natural conditions. In addition, I have used cultivation independent approach and tried to address the question of “which methanotrophs are actually there and doing the job of methane oxidation in sediment of Lake Constance”.

Table of Contents

1 General Introduction

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Methane Methanotrophs or Methane Oxidizing Bacteria (MOB) Phylogeny of Methanotrophs Overview of genera Aerobic Methane Oxidation Enzymes for Methane Oxidation Methanotrophic communities in Lakes Cultivation-Independent Approaches Cultivation of methanotrophs Anaerobic Oxidation of Methane (AOM) Perspective and Concept of this thesis

1 2 3 4 5 7 9 14 20 22 24

2. Cultivation of methanotrophic bacteria in opposing gradients of

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methane and oxygen Abstract Introduction Materials and Methods Results Discussion

25 26 28 36 47

3. Characterization of a novel methanotroph, Methylosoma difficile gen.

55

nov., sp. nov., enriched by gradient cultivation from littoral sediment of Lake Constance Summary Introduction Methods Results and Discussion

55 57 58 64

4. Comparison of aerobic methanotrophic communities in littoral and

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profundal sediment of Lake Constance by a molecular approach Abstract Introduction Materials and Methods Results Discussion

74 75 76 78 82

5. Depth distribution of methanotrophic bacteria in littoral and

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profundal sediments of Lake Constance Abstract Introduction Materials and Methods Results Discussion

85 86 88 94 103

6. Oxygen Relationships and Ultra-structure of a novel methanotroph

112

Abstract Introduction Materials and Methods Results Discussion

112 113 115 118 126

7. General Discussion

134

8. Summary

152

9. Zussamenfassung

154

References

157

Contributions to this thesis

170

Appendix

171

A Diatom associated bacteria and consumption of diatom derived EPS:

172

a study from epilithic biofilms in Lake Constance Abstract Introduction Materials and Methods Results Discussion

172 173 175 179 185

Chapter 1 : Introduction

Chapter 1 Introduction Methane Methane gas is produced by both natural and anthropogenic sources and it is 26 times more effective as a greenhouse gas as compared to CO2 (Lelieveld et al., 1993). The discovery of methane gas goes back to 1776, a gas which was collected from stirred sediment and which ignited readily (Dalton, 2005). Methane gas has a strong absorbance of infrared radiations which are not able to escape from the Earth’s atmosphere, leading to global warming. The concentration of methane has increased from 0.75 – 1.75 ppm in the last 300 years at an enormous rate and might reach 4.0 ppm till 2050 (Ramanathan et al., 1985). The release of methane to the atmosphere results in an increase in global warming and causes changes in the chemical composition of the atmosphere (Lelieveld et al., 1993). It has been predicted that increased levels of methane in the atmosphere will decrease OH radical concentrations and thus increase the lifetime of methane in the atmosphere (Lelieveld et al., 1993) and finally would result in decrease in the tropospheric ozone concentrations. The major natural and anthropogenic sources of methane include natural wetlands, paddy fields, ruminants, termites lakes and oceans, landfills, oil recovery operations, and methane hydrates (Dalton, 2005). Exactly 100 years ago, in 1906, the dutch microbiologist N. L. Sohngen isolated from pond water and aquatic plants a first bacterium growing on methane,

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Chapter 1 : Introduction and named it Bacillus methanicus (Sohngen, 1906). This organism was unfortunately lost and not many methanotrophs were discovered until 1950 -1960s. In 1970, Whittenbury and his coworkers (Whittenbury et al., 1970) started a new era of research on methanotrophs by isolating more than 100 strains which enabled the scientists to investigate their biochemical properties and pathways of aerobic methane oxidation in much detail. Methanotrophs or Methane Oxidizing Bacteria (MOB) Methanotrophs or Methane Oxidizing Bacteria (MOB) are a subset of methylotrophs (Hanson & Hanson, 1996) and have the unique ability to use methane as the sole source of C and energy. Methylotrophs are capable of utilizing a wide variety of C-1 compounds including methane, methanol, methylamines, etc. MOB are specialized, as they utilize mainly methane and sometimes also methanol as their sole source of C and energy, and use molecular oxygen as the terminal electron acceptor (Hanson & Hanson, 1996). Methylotrophs are phylogenetically widespread and include archaea, eubacteria and yeasts. On the other hand, known MOB belong only to the α and γ subclass of Proteobacteria. With only one proven exception, i.e. the genus Methylocella, MOB are obligately methylotrophic and cannot utilize complex C compounds such as sugars or organic acids (Bowman, 2000). The evolutionary and physiological reasons for this, remain speculative (Dedysh et al., 2005). In Type I methanotrophs this could be due to the lack of certain enzymes in the Krebs cycle like α ketogulatarate dehydrogenase. In the recently published genome of Methylococcus capsulatus it was shown that the genes coding all the necessary enzymes of the TCA cycle were present, but still this methanotroph cannot utilize organic compounds. Another reason could be the lack of specific transporters for complex C compounds (Dedysh et al., 2005). 2

Chapter 1 : Introduction Phylogeny of Methanotrophs Whittenbury (Whittenbury et al., 1970) and his colleagues derived a classification scheme for methanotrophs based on morphology, arrangement of intracytoplasmic membranes, pathway of C assimilation, nitrogen fixation ability, presence of cysts or spores, colony color and motility, and grouped them into five genera, i.e. Methylosinus, Methylocystis, Methylomonas, Methylobacter and Methylococcus. Using the following criteria, i.e. - numerical taxonomy, DNA-DNA hybridization, PLFA analysis, physiological properties and phylogenetic relationships, Bowman and his colleagues (Bowman et al., 1993) (Bowman et al., 1995) reorganized the phylogeny of methanotrophs. According to their scheme, the family Methylococcaceae (composed of Type I and Type X methanotrophs) should contain Methylococcus (Type X) and Methylomonas, Methylobacter, Methylomicrobium (Type I). The family Methylocystaceae (Type II methanotrophs) comprised the closely related groups Methylosinus and Methylocystis. After these rearrangements in phylogeny, seven more genera were added till 2006, which include Methylocaldum (Type X), Methylosphaera, Methylosarcina, Methylothermus, Methylohalobium (Type I), Methylocella and Methylocapsa (Type II). Type I methanotrophs have intracytoplasmic membranes arranged in bundles of vesicular discs, 16-carbon phospholipid fatty acids, and assimilate formaldehyde by the ribulose monophosphate (RuMP) pathway. Type II methanotrophs are characterized by assimilation of formaldehyde by the serine pathway, they have 18carbon phospholipid fatty acids and paired intracellular membranes aligned to the periphery of the cell. The membrane arrangements of the type II genera Methylocella and Methylocapsa are, however, different from the other Type 2 methanotrophs. The 3

Chapter 1 : Introduction characteristics of methanotrophs with respect to their phylogeny, membrane arrangements and phospholipid fatty acids are the key characteristics in the identification of methanotroph isolates obtained from the environment (Bowman, 2000). Overview of genera A total of 13 genera of methanotrophs have been described so far : Methylobacter, Methylomonas, Methylomicrobium, Methylosarcina, Methylothermus, Methylohalobium; and Methylosphaera belong to the Type I ; Type X have two genera Methylococcus, Methylocaldum, and Type II MOB include Methlyosinus, Methylocystis, Methylocella and Methylocapsa. Among the newly isolated genera, most are extremophilic. Methylocapsa and Methylocella (Dedysh et al., 2000) are acidophilic, Methylocaldum (Bodrossy et al., 1997) and Methylothermus are thermotolerant and thermophilic, respectively, Methylosphaera (Bowman et al., 1997) is psychrophilic and Methylohalobius is halophilic. Crenothrix polyspora : a well known bacterium, is a methanotroph!! Crenothrix polyspora, a well known conspicuous filamentous bacterium with a complete lifecycle was discovered 135 years ago by Ferdinand Cohn (the founder of Bacteriology). Recently it has been discovered to be a methane oxidizer with an unusual methane mono-oxygenase (Stoecker et al., 2006). This bacterium is infamous for mass development in drinking water systems and wells and remained uncharacterized till date. Characterization of 16S rDNA, methane-utilizing capacity and presence of pmoA sequences supported that it is a methanotroph.

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Chapter 1 : Introduction Aerobic Methane Oxidation

Fig.1. Aerobic oxidation of methane. Abbreviations :FADH-formaldehyde dehydrogenase. FDH-formate dehydrogenase, CytC-cytochrome c. Taken from: (Hanson & Hanson, 1996)

Pathways for the oxidation of methane and assimilation of formaldehyde The first step in aerobic methane oxidation (Fig.1) takes place with the help of specialized enzymes known as methane mono-oxygenases (MMOs). These enzymes break the O-O bond in the dioxygen molecule by utilizing two reducing equivalents (Hanson & Hanson, 1996). One of the oxygen atoms is converted to water and the other one is incorporated into methane to form methanol. Methanol is further oxidized to formaldehyde by methanol dehydrogenase (MDH). Formaldehyde is the central metabolite in the anabolic and catabolic pathways. In the catabolic pathway, HCHO is further converted to formate and then to CO2. HCHO assimilation takes place by two different pathways in Type I and Type II methanotrophs. In Type I methanotrophs, it takes place by the ribulose monophosphate (RuMP) pathway for assimilation of formaldehyde. 5

Chapter 1 : Introduction The pathway is shown below: (Fig. 2)

Fig.2. RuMP pathway for HCHO assimilation in Type I methanotrophs Taken from (Hanson and Hanson 1996).

The Type II methanotrophs use the serine pathway for assimilation of HCHO.

Fig.3. Serine pathway for assimilation of HCHO in Type II methanotrophs Taken from (Hanson and Hanson 1996).

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Chapter 1 : Introduction Enzymes for Methane Oxidation Two methane mono-oxygenases are known to be present in methanotrophs, i.e. soluble and particulate (sMMO and pMMO). Almost all known methanotrophs possess pMMO except Methylocella, whereas the soluble MMO is not present in all methanotrophs. The sMMO utilizes NADH + H + as the electron donor and is a nonheme, iron-containing enzyme complex consisting of three components: hydroxylase (245 kDa in size), protein B, a regulatory protein and C is the reductase component. The hydroxylase has three subunits, α, β and γ, of 60, 45 and 20 kDa respectively, which are arranged in a α2β2γ2 configuration. The α subunit contains a non-heme centre at the active site of the enzyme, where methanol is formed from methane and oxygen. The crystal structure of this enzyme is known and contains a carboxylatebridged di-iron centre (Lieberman & Rosenzweig, 2005). The genes encoding sMMO have been cloned and sequenced from several methanotrophs, including Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. In methanotrophs, these genes are clustered on the chromosome. mmoX, mmoY and mmoZ encode the α, β and γ subunits of the hydroxylase, respectively; mmoB and mmoC code for protein B and protein C , respectively. This enzyme has a broad substrate specificity and can co-oxidize a number of other aromatic compounds and hydrocarbons (Hanson & Hanson, 1996). pMMO is expressed only when the copper supply in the culture medium is high (Prior & Dalton, 1985). All known methanotrophs except Methylocella palustris harbor pMMO (Dedysh et al., 2005). Unlike sMMO, pMMO has relatively narrow substrate specificity and can oxidize alkanes and alkenes of up to five carbons in length. However, it could be useful for biotransformation as well (DiSpirito et al., 1992). pMMO is composed of three subunits with molecular masses of 47 kDa (β, 7

Chapter 1 : Introduction pmoB subunit), 26 kDa (α, pmoA subunit) and 23 kDa (γ, pmoC subunit), encoded by pmoA, pmoB and pmoC genes, respectively (Semrau et al., 1995). Most of the biochemistry, structure and mechanism of this predominant methane monooxygenase had remained unanswered in spite of the numerous efforts done in the last 20 years, until the crystal structure of pMMO was obtained (Lieberman & Rosenzweig, 2005). The crystal structure of pMMO from Methylococcus capsulatus (Bath) was obtained to a resolution of 2.8 Aº. The enzyme is now known to be a trimer with α3β3γ3 polypeptide arrangement. Two metal centres modeled as mononuclear copper and dinuclear copper, are located in the soluble regions of each pmoB subunit, which resembles cytochrome c oxidase subunit II. A third metal centre, occupied by zinc in the crystal (coming from the buffer used), is located in the membrane and it is not clear what could be there in the original structure in the place of zinc. There are two nearly identical copies of the genes encoding pMMO (pmoCAB) in the chromosome of Methylococcus capsulatus (Bath) (Semrau et al., 1995; Stolyar et al., 1999) and a third, separate copy of pmoC has also been identified (Stolyar et al., 1999). Comparison of pMMO and ammonia monooxygenase (AMO) gene sequences suggests that pMMO and AMO could be evolutionarily related (Holmes et al., 1995). The pmoA, which encodes the α subunit of pMMO, has been shown to be evolutionary highly conserved among methanotrophs (Holmes et al., 1995). In methanotrophs such as Methylosinus trichosporium OB3b, which possess both pMMO and sMMO, there is a metabolic switch mediated by copper ions. When cells are starved for copper, and the copper-to-biomass ratio of the cell is low, sMMO is expressed. Cells grown under conditions of excess copper express pMMO and there is no detectable sMMO expression (Murrell et al., 2000).

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Chapter 1 : Introduction Habitats Methanotrophs are ubiquitous, i.e. present in nearly all samples of swamps, sediment, rice paddies, oceans, ponds, soils from meadows, deciduous forest, streams, sewage sludge, etc. Wetlands and rice paddies are important sources of methane and oxic layers of sediments and soils act as biofilters for methane which is produced in the anoxic soil or sediment (Hanson & Hanson, 1996). Methanotrophic communities in Lakes Freshwater lakes contribute to a major portion, i.e. from 6 to 16 % of the global methane emissions, then thought before. Methane oxidation in lakes is an important process to prevent the escape of the methane produced in deeper anoxic sediment layers (Bastviken et al., 2004; Rudd & Hamilton, 1978; Thebrath et al., 1993) to the atmosphere, and eventually controls global warming. In mesotrophic or oligotrophic lakes which are oxic down to the sediment, aerobic methane oxidation occurs at the sediment-water interface (Frenzel et al., 1990; Lidstrom & Somers, 1984). The availability and rate of production of methane, as well as the availability of oxygen and nitrogen, determines the location and rates of methane oxidation (Hanson & Hanson, 1996). In stratified, eutrophic lakes, methane oxidation occurs near the bottom of the chemocline (metalimnion), where dissolvedoxygen levels are relatively low compared with levels in the epilimnion during summer stratification. Very little methane is found in the oxygenated epilimnion, and most of the methane produced in the anoxic sediments is stored in the hypolimnion, which is also devoid of oxygen, or is oxidized in the metalimnion (Hanson & Hanson, 1996). Methane oxidation in freshwater lakes was considered exclusively to be an aerobic process (Kuivila et al., 1988), (Eller et al., 2005a), (Frenzel et al., 1990) but 9

Chapter 1 : Introduction recently in Lake Pluβsee (Eller et al., 2005b), also anaerobic methane oxidation has been described. Lake Washington Lake Washington is a monomictic, mesotrophic freshwater lake close to Seattle. Different aspects of methane oxidation (Kuivila et al., 1988; Lidstrom & Somers, 1984) as well as methanotrophic and methylotrophic communities have been studied in the profundal sediment of this lake (62 m deep site) (Auman et al., 2000; Auman & Lidstrom, 2002; Costello & Lidstrom, 1999; Costello et al., 2002; Kaluzhnaya et al., 2006; Nercessian et al., 2005). Cultivation-independent studies were focused on the natural communities of MOB where maximal methane oxidation occurred, i.e. the top 1 cm (Costello & Lidstrom, 1999). Later, both Type I and Type II MOB were isolated from this layer (Auman et al., 2000). Further, the abundances of methanotrophic bacteria were determined by slot blot hybridization with Type I, Type II 16S rDNA and pmoA gene probes, PLFA (phospholoipid fatty acid) analysis, and methane oxidation rates (Costello et al., 2002). This was the first report where three independent quantitative studies were done with methanotrophs which were estimated to be about 108 to 109 cells per g dry sediment. Type I methanotrophs were found to be dominant, and at least one order of magnitude higher than Type II. Moreover the sMMO containing Methylomonas sp. dominated the whole community (Auman & Lidstrom, 2002). Thus, a particular type of methanotroph could be dominant in a particular habitat, based upon its characteristics. The peak of methane oxidation was located in the top 7-8 mm (Kuivila et al., 1988), although the methane oxidation potential in 0.5 cm sections of the top 1.5 cm showed similar methane oxidation potentials (Auman et al., 2000) in all three layers,

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Chapter 1 : Introduction suggesting that the number of methanotrophs in these layers did not differ significantly. Recently, in order to identify the communities active in metabolism of different C1 compounds (methanol, methylamine, HCHO and formate), two novel approaches were used, analysis of mRNA and rRNA by using RT (reverse transcription) PCR-based technique and DNA-SIP (Stable Isotope Probing) (Nercessian et al., 2005). Lake Constance Lake Constance is a pre-alpine lake and the second largest one in Europe. Various aspects of methane oxidation in Lake Constance have been studied in great detail over the past years (Bosse et al., 1993; Bussmann et al., 2004; Bussmann, 2005; Bussmann et al., 2006; Frenzel et al., 1990; Pester et al., 2004; Schulz & Conrad, 1995). Different from the profundal sediment, the littoral sediment is subject to changing environmental factors such as wind and waves, seasonal fluctuations of water level and temperature, and day-night cycles of light, and oxygen. Aerobic methane oxidation in the surface layer of the profundal sediment (147 m) of Lake Constance has been studied (Frenzel et al., 1990). It was found that 93% of the total methane was consumed under aerobic conditions and this contributed to around 9% of total O2 consumption. Further, it was found that phytoplankton blooms are responsible for seasonal variation in the concentration of the acetate, as the dominant substrate for methanogenic archaea (Schulz & Conrad, 1995). Another study was done to investigate activity and survival of bacteria from the deeper parts of the lake (Rothfuss et al., 1997). In this study, cultivable methanotrophic bacteria were found down to 32 cm depth but spontaneous methane oxidation (without induction with methane) was observed only in the upper 7.5 cm layer of the sediment. The deeper layers i.e. below 7.5 cm were active in methane oxidation after induction by

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Chapter 1 : Introduction incubation with methane in the headspace. Thus, it was proven that the methanotrophic bacteria might have survived many years of anoxic conditions and methane starvation. The role of spores or cysts has been predicted to be very important in this situation. In the littoral sediment of Lake Constance, CH4 production rates varied from 5 mmolm-2d-1 in December to 95 mmolm-2d-1 in September (Thebrath et al., 1993). Maximum amounts of CH4 were produced in the littoral zone in 2 – 5 cm depth (Thebrath et al., 1993), a large part of methane was found to be lost by ebullition. Methane oxidation in the littoral sediment was recorded in the surface layers of the sediment (0-1.5 cm); although a large methane oxidation potential was also found to be in the deeper layers down to 5 cm. The inhibitory effect of ammonium ions on the surface sediment from the littoral zone of Lake Constance was observed (Bosse et al., 1993). Recently, the effect of sediment resuspension by waves or bioturbation was studied in the littoral sediment and was found to be a major reason for methane release through the sediment (Bussmann, 2005). Methane profiles were recently recorded using a new methane sensor (Bussmann & Schink, 2006) based on (Rothfuss & Conrad, 1994). This method has enabled us to measure methane profiles with more precision. In littoral sediment of Lake Constance, MOB have been investigated by both culture-independent and cultivation-dependent methods. Here a stable and diverse community of both type I and type II MOB, and an apparent dominance of type I MOB could be documented with a T-RFLP and pmoA clone library approach (Pester et al., 2004). Attempts to optimise the cultivation conditions by modification of the composition of the medium and the gas atmosphere resulted in increased viable

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Chapter 1 : Introduction counts, but the diversity of the cultivated MOB still did not reflect the diversity of methanotrophs in this sediment (Bussmann et al., 2004). Mono Lake California Mono Lake, an alkaline salt lake located in California that undergoes seasonal stratification, is characterized by high salinity, and the methane is produced biogenically as well as thermogenically. Two studies were conducted to correlate the patterns of methane oxidation and associated methane oxidizing communities (Carini et al., 2005; Lin et al., 2005). A temporarily shifting zone of methane oxidation was consistently associated with a micro-oxic zone that migrated upwards in the water column as stratification progressed. Although a stable number of methanotrophs over depth and over time was observed, the change in methane oxidation zones were attributed to small shifts in the activity and ratios of the MOB communities. The study on the community structure was based on pmoA clone library and DGGE at 4 different depths. Methylobacter- related methanotrophs dominated the clone libraries, and Methylocystis appeared to be stratified at the zone of highest methane oxidation. Plußsee Plußsee is a eutrophic lake and has a stable thermal stratification in summer and anoxia in the hypolimnion (Eller et al., 2005b). 13C signature and methane, oxygen and sulfide profiles determined the zones of aerobic and anaerobic methane oxidation. The finding that aerobic and anaerobic methane oxidation occurs together was further supported by the detection of a relatively high number of aerobic MOB as well as a significant number of ANME in anoxic water column by fluorescent in situ hybridization (FISH) (Eller et al., 2005b). This seems to be the first report where anaerobic oxidation of methane (AOM) in a freshwater lake has been investigated.

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Chapter 1 : Introduction Other lakes and Chironomid Larvae A comparative study has been done on methane oxidation in two lakes in Northern Germany, i.e. Holzsee and Groβer Binnensee (Eller et al., 2005a). Stable isotope analysis showed that MOB could be an important food source for chironomid larvae because of the highly depleted 13C signatures. These two lakes are heavily inhabited by chironomid larvae. It was shown that the larval 13C depletion was not due to differences in the community structure but due to the activities and sizes of the MOB communities. Cultivation-Independent Approaches Due to the difficulties in cultivating methanotrophs (Bowman, 2000), various efforts have been undertaken to explore the methanotrophic diversity by cultivationindependent approaches. Mainly the 16S rDNA sequences of methanotrophs, and some of the functional genes, pmoA, mmoX and mxaF have been used as phylogenetic markers. The diversity of methanotrophs has been assessed by cloning, T-RFLP, DGGE, and micro-array approach. For quantification of methanotrophs, fluorescence in situ hybridization (FISH), use of PLFA (phospholipid fatty acid) profiles, real time PCR, etc. have been used. Stabe isotope probing (SIP), an attractive method to link bacteria to their functions, has been used also used extensively, in relation to aerobic methane oxidation. Community analysis: Clone Libraries In this method, a gene or a partial gene of interest is amplified from the DNA of an environmental sample and is cloned to generate a set of clones or a clone library. Molecular systematics of bacteria is based on the rRNA of the ribosomal small subunit. Thus, 16S rRNA or a functional gene is used for clone library 14

Chapter 1 : Introduction construction. Similar to DNA, RNA can be isolated, reverse transcribed and the cDNA clone libraries can be prepared. In case of methanotrophs, primers have been designed to specifically amplify partial 16S rDNA from Type I and Type II methanotrophs (Wise et al., 1999). Clone libraries of 16S rDNA and functional genes like pmoA (subunit A of pMMO), mmoX (α subunit of sMMO), mxaF (methanol dehydrogenase), etc. have been used to study methanotrophs from a variety of habitats including rice fields (Horz et al., 2001), landfills (Wise et al., 1999), freshwater lakes (Costello & Lidstrom, 1999), marine environment (Holmes et al., 1995) and soil (Knief et al., 2003). DGGE Denaturing gradient gel electrophoresis (DGGE) works by applying a small sample of DNA (or RNA) to an electrophoresis gel that contains a denaturing agent. Certain denaturing gels are capable of inducing DNA to melt at various stages. As a result of this melting, the DNA spreads through the gel and can be analyzed for single components. Sequence differences in otherwise identical fragments often cause them to partially melt at different positions in the gradient and therefore stop at different positions in the gel. TGGE (Temperature gradient gel electrophoresis) is based on the same principle, except it provides a temperature gradient instead of a chemical gradient. These are valuable fingerprinting techniques for studying microbial community structure. They separate a mix of PCR amplified gene fragments based on sequence differences and allow large number of samples to be analyzed simultaneously. They are particularly useful when, the dynamics of microbial communities is influenced by environmental changes. DGGE has been used for community analyses of 16S and

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Chapter 1 : Introduction functional genes of methanotrophs in rice fields (Henckel et al., 1999) and (Eller & Frenzel, 2001), in freshwater wetland communities (Bodelier et al., 2005). T-RFLP Terminal Restriction Fragment Length Polymorphism (T-RFLP) is a molecular biological technique initially developed for characterizing natural bacterial communities. The technique works by PCR amplification of DNA using primer pairs in which one of the primers is labeled with fluorescent tags. The PCR products are then digested using RFLP enzymes and the resulting patterns visualized using a DNA sequencer or a gel. The results are analyzed either by simply counting and comparing bands or peaks in the T-RFLP profile, or by matching bands from one or more TRFLP runs to a database (base clone library) of known species. The T-RFLP approach is similar to DGGE and can be used to monitor changes or differences within different communities. (Horz et al., 2001) used this technique for the detection of methanotrophic diversity on roots of rice, coupled with clone libraries of mxaF, mmoX and type I methanotrophs. The only disadvantage of this method is that it requires a base clone library for comparison and cannot be used like DGGE directly. This technique has been used to study methanotrophs from Lake Constance (Pester et al., 2004). It was useful for analyzing the differences in methanotrophic communities in the littoral and profundal sediment of Lake Constance, and in addition the temporal and spatial (depth wise) differences. This technique was also used for monitoring the methanotrophic community growing in a cultivation-based approach (Bussmann et al., 2004) and comparing the T-RFLP profiles of the growing bacteria to the original community in the littoral sediment of Lake Constance.

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Microarray approach The database of pmoA genes has been used to create microarray analysis of the methanotrophs. This technique requires a large amount of efforts in the initial establishment, but can be used as an efficient tool for analyzing the diversity of methanotrophs in new habitats. This microarray consisted of a set of 59 probes that covered the entire diversity of pmoA sequences available and could detect less dominant bacteria up to 5% of the total community (Bodrossy et al., 2003). Stable Isotope Probing (SIP) SIP is a technique that is used to identify the organisms in an environmental sample which use a particular substrate (Radajeweski et al., 2000) (Dumont & Murell, 2005). Thus, this method relates the identity of bacteria with their metabolic role in the environment. In this method, a substrate which is highly enriched in a stable isotope such as 13C is incorporated into the cellular components of the bacteria utilizing it. Usually DNA/RNA being the most informative biomarkers are used which can be separated by density gradient centrifugation. The nucleic acids are further analyzed by amplification of 16S rDNA or functional genes followed by cloning, or more advanced techniques like metagenomic analysis can be combined to unravel the metabolic pathways of the active bacteria. Methanol and methane were amongst the earliest and most ideal substrates used. SIP has been used for determining active methano- or methylotrophic populations in different habitats, like peat soil microcosms (Morris et al., 2002), Transbaikal soda lakes (Lin et al., 2004), in acidic forest soils (Radajeweski et al., 2002), freshwater lakes (Nercessian et al., 2005), Movile cave (Hutchens et al., 2004). The drawbacks of this method are 1. Longer incubation times can result in labeling those bacteria

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Chapter 1 : Introduction which are cross feeding on the intermediates and 2. High substrate concentrations can select for the fast growing bacteria. SIP coupled to Metagenomics Metagenomics is the study of genomes recovered from environmental sample. This relatively new field of genetic research allows the genomic study of organisms that are not easily cultured in a laboratory. SIP enables to link organisms in the environment to functions as described above. If the isolated heavy DNA is used for metagenomic analysis, i.e. isolation of large inserts from uncultivated bacteria, entire operons or set of genes can be analyzed from an environment. Recently, such an approach has been used for a forest soil sample, for which a prior SIP experiment was done (Radajeweski et al., 2002). In this study, an entire methane mono-oxygenase operon was found on an insert of a 15 kb clone (Dumont et al., 2006). Fluorsecence in situ hybridization-flow cytometry-cell sorting-based method (FISH-FACS) In this method FISH is carried out after extracting the cells from an environmental sample, followed by cell sorting. This method was used as a novel approach for the separation of Type I and Type II methanotrophs from the surface sediment of Lake Washington (Kaluzhnaya et al., 2006). This is a promising tool for genetic and genomic characterization of yet uncultivated and uncultured microbes. Further advances in this method could employ mRNA based cell sorting followed by whole genome amplification (WGA). Abundance distribution FISH Fluorescence in situ hybridization (FISH) has been a breakthrough for the phylogenetic, determinative and environmental studies in microbiology (Amann et al.,

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Chapter 1 : Introduction 1990). For identification and quantification of methanotrophs, FISH probes were designed by Eller et al (Eller et al., 2001). Although, before this study there were a few probes already existing which distinguished between γ proteobacterial and α proteobacterial methanotrophs (Holmes et al., 1995). In this study (Eller et al., 2001) a pair of probes for γ proteobacterial methanotrophs (Mγ705 and Mγ84).and a probe Mα450 for the detection of α proteobacteria was designed. These probes have fewer mismatches to the sequences of available methanotrophs and less false positive strains, and are most widely used. FISH has been used for detection and / or enumeration of methanotrophs from various habitats such as rhizoplane samples (Eller & Frenzel, 2001), in Sphagnum peat bogs (Dedysh et al., 2001) and in sediment of lakes and chirinomid larvae (Eller et al., 2005a). Lipid Biomarkers The use of specific phospholipids fatty acid biomarkers (PLFA) has been proven to be useful for determining the abundance of methanotrophs (Bowman, 2000). In methanotrophs, 16:1 ω8c is a characteristic biomarker used for Type I methanotrophs and 18:1 ω8c for Type II methanotrophs. The detection of signature fatty acids rely on the conformation of double bond geometry and isomeric state using gas chromatography coupled to mass spectrometry (GC-MS). This method lacks sensitivity, i.e., it is reliable only when the communities are (>1%) (Bowman, 2000). Real Time PCR Real Time PCR was developed basically for the quantitative determination of methanotrophs from soil because the MPN method or FISH are cumbersome and require large manual efforts (Kolb et al., 2003), whereas methods like lipid biomarkers have limited ability to distinguish between subgroups of methanotrophs. Since pmoA and 16S rDNA clusters in phylogenetic trees are congruent (Holmes et

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Chapter 1 : Introduction al., 1995) and pMMO is present in all methanotrophs except Methylocella. These assays were based on pmoA with a detection limit of 10-100 cells (Kolb et al., 2003). Recently, for quantification of aerobic and anaerobic methanotrophs from the black sea water column, real time PCR assays were developed specifically for Type I and Type II methanotrophs and ANME (anaerobic methane oxidizing archaea) (Schubert et al., 2006). Real time PCR assays have also been developed for unusual pmoA genes of Crenothrix polyspora (Stoecker et al., 2006) and for mmoX genes in Methylocella palustris (Dedysh et al., 2005).

Cultivation of methanotrophs Cultivation of methanotrophs goes back to around 100 years, when Sohngen isolated the first methanotroph. The real landmark in the cultivation of methanotrophs was when Whittenbury and his colleagues isolated more than 100 methanotrophs. Traditional methods Whittenbury et al (1970) mainly used microscopy to detect tiny colonies of methanotrophs on the plates which were uncontaminated with heterotrophs and obtained > 100 cultures of methanotrophs. They also developed two media for cultivation of methanotrophs and named them NMS (Nitrate Mineral Salt medium) and AMS (Ammonium Mineral Salt medium). Till date this has remained to be the classical method and the classical media for isolation of methanotrophs and has yielded many new strains. Small variations have been made in the methodology e.g. use of microtitre plates. Non-conventional cultivation methods Gradient cultivation Since methanotrophs are difficult to cultivate by classical plating-based methods, a method which mimics natural conditions for the methanotrophs was 20

Chapter 1 : Introduction developed –‘Gradient cultivation’. Here, methanotrophs are grown in opposing gradients of methane and oxygen in agarose diffusion columns (Amaral & Knowles, 1995). Gradient cultivation allows both substrates, i.e., methane and oxygen to diffuse, and bacteria can develop wherever these gradients meet and grow as a band. In this study, Type I methanotrophs were found at low methane and high oxygen concentrations (by hybridization studies) whereas Type II were found where methane concentrations were high and oxygen concentrations were low. When ammonium – based medium was used, nitrite was formed below the band creating toxic conditions. In nitrate-based medium, denitrifying conditions were found below the band creating anoxic conditions and supplying nutrients to denitrifying bacteria (Amaral et al., 1995). Thus, this method was basically designed to isolate novel methanotrophs which do not grow in conventional cultivation methods and offers some advantages with a possibility of chemical measurements, morphological description and nucleic acid studies. Although only one methanotroph has been isolated by this study, Methylobacter sp. T20 (Ren et al., 2000), this method actually formed the basis of our cultivation strategy. Soil Membrane Culture Method This is a new method for isolation of methanotrophs from soil (Svenning et al., 2003). Soil samples are plated on polycarbonate membranes which were incubated in a methane-air atmosphere using a non-sterile soil suspension as a medium. The membrane was permeable and the soil acted as a buffer absorbing the excess of metabolites excreted, allowing the growth of methanotrophs not allowing other contaminants to grow. Both Type I and Type II methanotrophs were cultivated by this study as revealed by FISH.

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Chapter 1 : Introduction Anaerobic Oxidation of Methane (AOM) Coupled to sulfate reduction Anaerobic methane oxidation (AOM) is the major sink of methane in marine sediments. AOM is assumed to be a reversal of methanogenesis coupled to reduction of sulfate to sulfide and is believed to involve methanogenic archaea (ANME) and sulfate reducers (SRB) as syntrophic partners (Boetius et al., 2000). The reaction is thought to be as followsCH4 + SO42- + H+= CO2 + HS- + 2H2O (Strous & Jetten, 2004) ANME archaea belong to three different groups and are known as ANME -, ANME-2 and ANME-3 although new groups also might be discovered, as newer habitats are surveyed. ANME-1 are distantly related to Methanosarcinales and Methanomicrobiales. ANME-2 group are more closely related to Methanosarcina and ANME-3 to Methanococcoides species. The SRB belong to the DesulfosarcinaDesulfococcus (DSS) group and are known to be in aggregates with ANME 2. SRB of the Desulfobulbus branch have been associated with ANME-3 aggregates (Lösekann, 2006). Even if AOM have been reported to occur in a variety of habitats, none of the partner bacterium have been isolated yet or cultivated, and the enzymes and biochemical pathways involved in AOM still remain unknown. Very recently, a candidate enzyme (Ni-protein) has been described that may catalyse the reverse methyl CoM reductase reaction (Krüger et al., 2003). ANME were first detected in sediments or cold seeps of various marine habitats, Black Sea sediment and hydrate ridge. The habitats for ANME 1 and 2 are different. ANME 1 are found in deep parts of the sediment in completely anoxic conditions and ANME 2/DSS clusters are found in slightly oxic parts also (Lösekann, 2006). 22

Chapter 1 : Introduction Coupled to nitrate reduction Very recently, it was discovered that AOM can be also coupled to denitrification (Raghoebarsing et al., 2006). This possibility was already discussed by (Strous & Jetten, 2004), until it was proven by Raghoebarsing and her colleagues that a microbial consortium can actually carry out this process. The microorganisms that couple the anaerobic oxidation of methane (AOM) to denitrification, is shown in equations (1) and (2).

(Raghoebarsing et al., 2006) The consortium was isolated from anoxic sediment from a canal in Netherlands, where the sediment was saturated with methane and nitrate concentrations were up to 1 mM. After 16 months of enrichment, this consortium was completely dominated by only two partners: a bacterium representing a new phylum having no cultured bacterium (80%) and an archaeon (10%).

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Perspective and Concept of this thesis The main aim of my thesis is to explore the aerobic methanotrophic communities dominant in the sediment of Lake Constance. Though my main focus is on studying the littoral sediment, for comparison purposes profundal sediment was also explored, as these two habitats share the same lake chemistry, but have different environmental conditions. I have used cultivation-based and cultivation-independent approaches to fulfill this purpose. Various aspects of gradient cultivation enrichment technique, which we used for enrichments of the methanotrophs from the littoral sediment, are addressed in Chapter 2. These include enrichment of methanotrophs followed by isolation and characterization of the methanotrophic communities in comparison with the methanotrophic communities in the sediment. Then, a description of a novel strain of methanotroph follows in Chapter 3. This novel Type I methanotroph has been obtained after an initial gradient culture enrichment and we describe it as a new genus and species of Type I methanotrophs (Methylosoma difficileT). In the following two chapters (Chapter 3 and 4) I describe the community structure and abundance of methanotrophs occurring in the littoral and profundal sediments by molecular approaches. The abundance of methanotrophs has been compared with the distribution of methane and oxygen in the sediments. In the last chapter (Chapter 6) I have studied some physiological and structural aspects of the new methanotroph described in Chapter 3 (Methylosoma difficile LC 2T). 24

Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen

Chapter 2 Cultivation of methanotrophic bacteria in opposing gradients of methane and oxygen Ingeborg Bussmann*, Monali Rahalkar, Bernhard Schink Published in FEMS Microbiology Ecology 56 (2006), 331-344

Abstract In sediments, methane-oxidizing bacteria live in opposing gradients of methane and oxygen. In such a gradient system, the fluxes of methane and oxygen are controlled by diffusion and consumption rates, and the rate-limiting substrate is maintained at a minimum concentration at the layer of consumption. Opposing gradients of methane and oxygen were mimicked in a specific cultivation set-up in which growth of methanotrophic bacteria occurred as a sharp band at either approx. 5 mm or approx. 20 mm below the air-exposed end. Two new strains of methanotrophic bacteria were isolated with this system. One isolate, strain LC 1, belonged to the Methylomonas genus (type I methantroph) and contained sMMO. Another isolate, strain LC 2, was related to the Methylobacter group (type I methantroph), as determined by 16S rRNA gene and pmoA sequence similarities. However, the partial pmoA sequence was only 86% related to cultured Methylobacter species. This strain accumulated significant amounts of formaldehyde in conventional cultivation with methane and oxygen which may explain why it is preferentially enriched in a gradient cultivation system. Key words: freshwater sediment, gradient cultivation, Lake Constance, methanotrophs, Methylobacter, Methylomonas, pMMO, sMMO

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Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen Introduction Methanotrophic or methane-oxidizing bacteria (MOB) are an important group of bacteria that use CH4 as their sole source of carbon and electrons. There is an increasing interest in MOB because of their importance in greenhouse gas consumption and their potential application in bioremedial degradation of industrial pollutants, e.g., trichloroethylene (Hanson & Hanson, 1996). MOB need both methane as electron donor and oxygen as co-reactant in the oxygenase reaction and as electron acceptor. In sediments, methane diffuses upwards from deeper sediment layers, and oxygen diffuses from the water column into the sediment. Both gases overlap at very low concentrations in the top few mm below the sediment surface where MOB can live in counter gradients of methane and oxygen. In this narrow zone, methanotrophic growth is limited by the diffusive transport of both substrates. MOB include species in the Alphaproteobacteria (type II MOB) and in the Gammaproteobacteria (type I MOB) (Bowman, 2000). The oxidation of methane to methanol is catalysed by either a soluble or a membrane-associated form of methane monooxygenase (sMMO and pMMO, respectively) (Hanson & Hanson, 1996). The pMMO genes are almost universal in MOB. One gene of this operon, pmoA, is strongly conserved and can be used as a functional phylogenetic marker for MOB in general (Holmes et al., 1995). In profundal sediment of Lake Washington, the enrichment of MOB with mineral medium (Whittenbury et al., 1970) led to the isolation of type I and type II MOB at almost equal numbers (5 and 6 strains out of 11, respectively). Two sMMO containing strains were isolated and assigned to the genus Methylomonas although this type of methanotrophs had not been reported before from a pristine environment (Auman et al., 2000). In a further study, it was shown that the major methanotrophic

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Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen population in Lake Washington sediment consists of sMMO-containing Methylomonas like MOB (type I) (Auman & Lidstrom, 2002). The number of type I MOB in this sediment, as estimated with cultivation-independent methods, is an order of magnitude higher than that of type II MOB (Costello et al., 2002). In littoral sediment of Lake Constance, MOB have been investigated by both culture-independent and cultivation-dependent methods. Here a stable and diverse community of both type I and type II MOB, and an apparent dominance of type I MOB could be documented with a T-RFLP and pmoA clone library approach (Pester et al., 2004). Attempts to optimise the cultivation conditions by modification of the composition of the medium and the gas atmosphere resulted in increased viable counts, but the diversity of the cultivated MOB still did not represent the diversity of methanotrophs in this sediment (Bussmann et al., 2004). Intermediates of methane oxidation such as methanol, formaldehyde, and formate have been detected in methanotrophic cultures, and they may reach even inhibitory concentrations (Agrawal & Lim, 1984; Costa et al., 2001). Production of formaldehyde and formate is favoured under unbalanced growth conditions if such bacteria are grown with methanol at high concentrations of oxygen. Removal of these intermediates, e. g., by a methylotrophic partner organism increases the methane oxidation rate of MOB (Wilkinson et al., 1974). Another way to avoid selfintoxication of MOB by possibly excreted toxic intermediates is the cultivation of these bacteria in counter gradients of methane and oxygen as this was first described by the lab of R. Knowles (Amaral et al., 1995; Amaral & Knowles, 1995). The aim of our present study was to combine the gradient technique and an optimized mineral medium (Bussmann et al., 2004) for cultivation of novel and ecologically relevant methanotrophs from littoral sediment of Lake Constance. The

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Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen diversity of MOB growing in the gradients was compared with the total diversity of MOB in Lake Constance sediment on the basis of pmoA clone libraries. Material and Methods Study site and sediment sampling Experiments were carried out with sediment from the lower infralittoral zone ("Litoralgarten", 47°41'N, 9°12'E) of Lake Constance, Germany. At the study site, CH4 concentrations in the sediment ranged from 20 to 90 µM at the sediment surface (Bussmann, 2005). The sediment consisted mainly of fine sand with a porosity of 0.62. Sediment cores (Ø 2.3 cm) were taken by SCUBA diving or with a sediment corer (∅ 8 cm) at 2–5 m water depth. Cultivation of MOB in liquid or on solid media Methanotrophs were grown in diluted mineral medium supplemented with 7 vitamin solution (Widdel & Pfennig, 1981) and were incubated at 16°C or 20°C in desiccators under an atmosphere of 17% O2, 24% CH4, 2% CO2 and 57% N2 (Bussmann et al., 2004). Solid media in plates contained 1.2% agarose. MOB were also grown in liquid medium in microtiter plates. For positive growth the OD595nm had to be 1.5 times more than the OD of a sterile control. To test for non-methanotrophic growth, cultures were streaked on plates with diluted complex medium (Bussmann et al., 2001) or Luria Bertani (LB) agar (Eisenstadt et al., 1984) and incubated without methane. Pure cultures of Methylobacter luteus type I and Methylosinus trichosporium type II (a gift by Peter Dunfield, MPI Marburg) were grown in liquid NMS medium (Whittenbury et al., 1970). Cultivation of MOB in gradients Bacteria were cultivated in glass tubes (inner ∅ 8 mm, length 12 cm) with screw caps at both ends. They were sealed with PTFE filters (TE 36, Schleicher & 28

Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen Schuell) that were supported by perforated silicone septa. Agarose (0.2% w/v; Agarose NEEO, Ultra Quality, Roth, Karlsruhe, Germany) was added to the diluted mineral medium to obtain a semi-solid consistency. Tubes were supplied with inoculum; then the anoxic and warm (38°C) medium was added and mixed immediately. The incubation chamber carried 42 gradient cultivation tubes and consisted of two chambers (6.5 l volume each) separated by an intermediate bottom which held the cultivation tubes in gas-tight rubber seals. The upper chamber was filled with air; and the lower one was flushed for 20 min (to exchange its volume 3 times) with 2% CO2, 24% CH4 and rest N2. The gas mixture was water saturated by passage through a washing flask to prevent evaporation from the tubes. The incubation temperature was either 16°C or 20°C. Tubes were checked once a week for presence of bands, and the gas mixture was renewed accordingly. To verify if the observed bands were due to growth of MOB, the distribution of oxygen and methane was determined. Dissolved oxygen was measured with a Clarktype microelectrode (Ox50, Unisense, Aarhus, DenmarK) at 1 mm intervals. Methane concentrations were determined with a methane sensor modified after (Rothfuss & Conrad, 1994). Isolation of methanotrophs Surface sediment (upper 1 cm) was used as inoculum. Two ml of sediment was mixed with 8 ml of mineral medium, and then further diluted (nine to ten steps). These dilutions (0.3 ml) were used as inoculum for 3 ml of diluted mineral medium in gradient tubes. The final dilutions ranged from 2 × 10-1 to 1 × 10-7. Usually 4-5 replicates were set up for each dilution step, along with controls without methane, without oxygen, and without inoculum. After band formation was observed, the tubes

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Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen were removed from the box, the agarose column was pushed out with a rubber plunger, and the bands were excised aseptically with a sterile scalpel. Bands from replicate tubes of the last positive dilution were pooled, resuspended in liquid mineral medium, vortexed and around 500 µl was used for inoculation of another 1:10 dilution series with liquid medium. After incubation for 2-3 weeks, the last positive dilution tube was used again for inoculation of another dilution series until finally one single morphotype dominated. The last three positive dilutions were streaked on plates and incubated with and without methane. A binocular microscope was used to pick smaller colonies which were resuspended and streaked on fresh plates until a pure culture was obtained. Isolates were checked frequently for non-methanotrophic contaminants after streaking on complex media. The cultures were examined with a phase contrast microscope (Axiophot; Zeiss, Oberkochen, Germany) and photographed using a cooled charge-coupled device camera (Magnafire, INTAS, Goettingen, Germany). Isolates were maintained at 4°C under a methane atmosphere for longer storage.

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Alcian Blue staining Polysaccharides produced after growth, in liquid or on solid medium or in gradients for three weeks were stained with Alcian blue (Hilger et al., 2000 ). Cell material was scraped from plates, liquid cultures were used directly, cell material from gradient cultures was cut from bands and suspended in ca. 300 µl of liquid mineral medium. Twenty µl of 1% Alcian blue solution in ethanol was diluted 1:10 with deionised water and mixed with ca. 20 µl of sample. Negative controls were prepared with pure agarose, to check for staining of agarose. Chemical analyses Formaldehyde was analyzed in the gas phase of incubation vessels by gas chromatography. Standards were prepared in glass tubes closed with butyl rubber stoppers. Formaldehyde standards were prepared from a fresh 37% (w/v) formaldehyde solution (Merck, Darmstadt, Germany) ranging from 0.01% to 2% (v/v). The formaldehyde concentration in the gas phase was estimated according to (Grützner & Hasse, 2004), gas phase- liquid equilibrium were calculated according to (Flett et al., 1976). DNA extraction and PCR amplification DNA was extracted from cell material, by a combination of enzymatic lysis (Ohkuma & Kudo, 1996) and bead beating (Henckel et al., 1999) with the following modifications: Cell material from gradient culture bands (200-500 µl) was used for DNA extraction. In case of plates, colonies were scraped or cell pellets were obtained from 1 - 2 ml of liquid cultures after centrifugation for 10 min at 13,000 rpm, 4 °C. Cell material was suspended in 800 µl buffer (100 mM Tris HCl, pH 8.0, 50 mM EDTA) and homogenized with plastic pestles (Micropistill sticks, Eppendorf,

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Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen Hamburg, Germany). The homogenates were transferred into screw-cap tubes with 0.7 g silica beads (0.1 mm diameter) and lysozyme (5 mg / ml). After incubation for 20 min. at 37°C, proteinase K (100 µg / ml) was added and the mixture was incubated again at 37°C for 40 min. After bead-beating (6.5 m/s, 45 s), proteins and debris were removed by two times washing with chloroform:isoamyl alcohol (24:1 v/v) in phase lock gel tubes (Eppendorf). The DNA was finally precipitated with 0.7 volume of isopropanol and harvested by centrifugation at 20800 g for 60 min, followed by removing the salts with 70% (v/v) ethanol and drying. The DNA was resuspended in c. 50 µl 10 mM Tris-EDTA buffer and stored at -20°C. DNA from pure cultures was used for amplification of 16S rRNA genes, using 27f (Edwards et al., 1989)and 1492r (Weisburg et al., 1991) universal primers. DNA from gradient cultures and pure cultures was used for the amplification of partial pmoA gene, using the pmoA primer pair A189f-mb661r (Costello & Lidstrom, 1999), additionally with isolates, amplification of partial pmoA gene was also checked with primer pair pmoA A189fA682r (Holmes et al., 1995). For amplification of the mmoX gene, primers mmoXA and mmoXB were used. For the construction of a sediment pmoA clone library littoral sediment (upper 1cm layer) was collected in August 2005. DNA was extracted with the using PowerSoil™ DNA Isolation Kit (MO BIO Laboratories, Inc., Solana Beach, CA). All amplifications were carried out in 50 µl or 25 µl total volume in an Eppendorf thermal cycler using recombinant Taq DNA polymerase (Eppendorf) or FailSafe™ Enzyme Mix and Premix B (Epicentre, Madison, WI) for clone library construction. For amplification of 16S rRNA genes, an initial denaturation at 94°C for 3 min was done, followed by 32 cycles at 94°C for 30 sec, 53°C for 30 sec and 72°C for 1 min, with a final extension step at 72°C for 10 min. For amplification with Type

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Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen I and Type II methanotroph specific primers (Wise et al., 1999) as well as for partial pmoA and mmoX genes, the following program was used: Initial denaturation at 94°C for 3min, followed by 32 cycles at 94°C for 30 sec, 56°C for 1min and 72°C for 90 sec, followed by final extension at 72°C for 10 min. PCR products were checked for amplification on 1.5% agarose gel by electrophoresis. Clone libraries and restriction fragment length polymorphism (RFLP) pmoA clone libraries from sediment and from gradient culture bands were prepared by cloning the partial pmoA gene product (508 bp), obtained after amplification with primers A189f-mb661r (Costello & Lidstrom, 1999) and purification of the PCR products. PCR products were purified using the Qiagen PCR purification kit (Qiagen, Hilden, Germany). All cloning steps were done using TA cloning kit (Invitrogen). In case of libraries with gradient culture bands, thirty clones from each clone library were selected randomly and were subjected to tooth pick PCR, using primers A189f-mb661r. The amplified products were digested with Msp I (5 U, MBI Fermentas), separated on 3.5% Nu-Sieve agarose (NuSieve ® 3:1 Agarose, Cambrex Bio Science Rockland Inc., ME), grouped according to their restriction patterns and each clone was assigned to an operational taxonomic unit (OTU) which represented a unique RFLP. Sediment clone libraries were analysed similarly, except more clones i.e. around seventy clones were randomly picked to cover the entire diversity and were digested with MspI/HaeIII (5 U, MBI Fermentas) and grouped as described above. 16S rRNA gene clone libraries were performed with DNA extracted from gradient bands. DNA was PCR amplified with the universal bacterial primers 27f (Edwards et al., 1989) and 1492 r (Weisburg et al., 1991) and cloned separately as

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Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen described above. Clones were digested with MspI restriction enzymes, RFLP analysis was done and OTUs were assigned as described above. Cloning, sequencing, and phylogenetic analysis With our isolates strain LC 1 and LC 2, complete 16S rRNA gene sequences were obtained by cloning the fragments using the TA cloning kit (Invitrogen). Clones were sequenced with primers 27f (Edwards et al., 1989), 533f (Lane et al., 1985), 1492r (Weisburg et al., 1991), MethT1dR (Wise et al., 1999) and assembled using the DNAStar software (http://www.dnastar.com). Similarly, sequences of the partial pmoA gene of the isolates and the partial mmoX gene of strain LC 1 were obtained by direct sequencing of the PCR products, and a complete sequence was obtained after cloning the fragment using TA cloning kit and sequencing from both ends. In case of pmoA and 16S rRNA gene clone libraries, representative clones from each OTU group were sequenced. At least 10% of clones from each RFLP group were sequenced. pmoA and mmoX clones were sequenced using M13f and M13r primers. Representative clones from 16S rRNA gene clone library were either sequenced completely with 27f (Edwards et al., 1989), 1492r (Weisburg et al., 1991) (clones representing dominant OTU groups) or partially with 27f primer (clones which were less frequent). All sequencing reactions were done at GATC Biotech AG (Konstanz, Germany). Blast search was performed at the NCBI site (http://www.ncbi.nlm.nih.gov/) (Altschul et al., 1990) and closely related sequences were retrieved. All sequences were checked for chimeras by dividing the sequence in two partial sequences and performing blast search. Two chimeras were found in 16S rRNA gene clone libraries.16S rRNA gene sequences of strain LC 1 and strain LC 2 were phylogenetically analysed using the ARB software package (version 2.5b; http://www.arb-home.de) (Ludwig et al., 2004). The new sequences were added to the

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Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen ARB database and aligned using the FAST Aligner tool as implemented in ARB. Alignments were checked and manually corrected where necessary. Sequences with more than 1400 nucleotides were used for alignment. Only those positions which were identical in 50 % of all sequences were used to create a filter. Phylogenetic analysis was done using the maximum likelihood, neighbour-joining and maximum parsimony algorithms as implemented in ARB. Phylogenetic distances were determined by calculating the similarity matrix within ARB using E.coli 16S rDNA genes as filter. For phylogenetic analysis of pmoA gene sequences they were translated within ARB to obtain deduced amino acid sequences and phylogenetic distance dendrograms were constructed using different methods such as Neighbour-joining, Desoete, and PHYLIP with the Fitch and Margoliash method (Felsenstein, 1989), using representative sequences of pmoA clones and isolates obtained in earlier studies done on Lake Constance as well as other studies (Pester et al., 2004). All sequences have been deposited in GenBank under accession numbers DQ119042- DQ119051 (sequences from gradient clones and isolates) and DQ235456- DQ235470 (sequences from sediment pmoA clone library).

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Results Growth of MOB in gradient cultures Initial experiments had shown that gradients of oxygen (and we assume also methane gradients) establish within 3 - 4 days in a 4.5 cm agarose column. Enrichment cultures from sediment developed 0.5 mm thick bands of bacterial growth after 2 -3 weeks of incubation. A narrow, homogeneous band, rather than single colonies, was taken as indication of methanotrophic growth in gradient tubes, as described in (Amaral & Knowles, 1995). If methane was excluded from the tubes such bands were never observed. Bands occurred typically approx. 5 mm below the air-exposed end, ranging from 2 – 5 mm. In some cases, a thin band was observed at approx. 20 mm below the air-exposed end, ranging from 20 – 25 mm. In older enrichment culture tubes (> 4 weeks) further bands developed like shadows below the first band. The distribution of oxygen and methane in these gradients was also measured (Fig. 1.). Oxygen penetrated only to the depth of the growth band (5 or 20 mm), and methane was not detectable above the bands. In a sterile control tubes, oxygen penetrated much deeper into the column and methane reached up to the surface. Thus, the growth bands were always observed where the concentrations of oxygen and methane approached zero.

36


Subsequent transfers of the bands into new gradient tubes were often not successful. After the 4th transfer, growth or bands could not be observed anymore. To check for possible reasons for this failure we performed various experiments. Enrichments in the gradient tubes were started with the medium optimised for MOB growing in liquid cultures. Therefore we checked if the MOB growing in the gradient tubes prefer a different medium composition. Cell material from two to three 37

Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen bands from initial sediment enrichments was pooled, and aliquots were transferred into the same medium again or into modified media. After incubation for three weeks, tubes were checked for growth bands. Each medium modification was tested three times. Increasing the phosphate and nitrate content (150 µM K-Na-PO4, 50 µM NO3) to 2-, 5- or 10-fold did not result in better growth. Addition of organic supplements (7 vitamin solution, 0.05% yeast extract, or 0.05% prefermented yeast extract), different buffers (0.01 M MOPS, 0.01 M TES or 0.01 M K-Na-PO4) and different mineral composition (standard medium, full-strength medium according to Widdel (Widdel, 1988), medium according to Whittenbury (Whittenbury et al., 1970)or (Heyer et al., 1984) had no influence on growth of transferred cultures. Cultures of MOB that had been cultivated always in liquid or solid medium were checked for growth in gradient cultures. Exponentially growing liquid cultures of Methylobacter luteus and Methylosinus trichosporium were inoculated i) into freshly prepared liquid medium with warm agarose, ii) into already solid medium in tubes stored under nitrogen, iii) on the surface of solid medium with a 3 day-old gradient, or iv) 5 mm below the surface of solid medium with a 3 day-old gradient. Both strains grew in the gradient tubes. Bands looked best (sharp, homogenous) when inoculated into freshly prepared medium. In an additional experiment, we tested how many cells of MOB were necessary to form a band in gradient tubes, compared to formation of turbidity in a microtiter plate. An exponentially growing culture of M. trichosporium was counted microscopically and diluted in 1:2 steps down to 3 cells per ml. Aliquots were transferred into gradient tubes (3 ml) and into microtiter plates (240 µl) resulting in the same cell number per vial. Gradient tubes were incubated in the incubation chamber and the microtiter plates in a desiccator, both with 3 parallels per dilution

38

Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen step. No growth was observed anymore with less than 7 cells per gradient tube (= 2 cells ml-1) and less than 2 cells per microtiter well (= 9 cells ml-1). To estimate the number of MOB in the sediment MPN-counting was done in gradients tubes and in liquid culture. For the gradient tubes 5-10 dilution steps with 34 parallels each and for the liquid cultures 12 dilutions with 8 parallels each were used (Bussmann et al., 2004). This experiment was repeated twice. At both sampling dates highest counts were obtained with liquid cultures in microtiter plates, 1.2 and 5.1 x 104 cells per ml respectively. The number of MOB obtained in gradient tubes reached only 3.6 x 103 cells per ml, at both dates. Isolation of methanotrophs from gradients Isolation of MOB from gradient enrichment cultures by streaking on plates led mostly to type I MOB. We describe here some of the strains isolated by gradient cultivation technique. Lake Constance Isolate 1 (Strain LC 1) Strain LC 1, a type I methanotroph, was isolated from a gradient enrichment culture obtained from a 1.6 × 10-3 dilution of the sediment. Strain LC 1 was isolated on solid medium, but it grew also in gradient culture and formed a band usually approx. 5 mm below the air-exposed end (Fig. 1a). LC1 is a motile, fat rod and a type I MOB. Phylogenetic analysis on the basis of the 16S rRNA gene sequence indicated that strain LC 1 is closely related to Methylomonas methanica (99.4%) and Methylomonas sp. LW15 (99.5%) isolated from Lake Washington (Fig. 2). The interesting feature of the strain is that the partial pmoA gene cannot be amplified with the pmoA primer A189f-A682r, but only with primer A189f-mb661r. A weak band is obtained at lower annealing temperatures if primer A189f-A682r is used. The sequence of the product obtained by A189-mb661 shows 95 % nucleotide identity

39

Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen with the pmoA sequence of Methylomonas sp. LW 15 (Fig. 3). Since strain LC 1 is closely related to Methylomonas sp. LW15, it was tested for the presence of sMMO with the primers mmoXA and mmoXB (Auman & Lidstrom, 2002). Strain LC 1 showed a positive PCR product of correct length (1230 bp). The sequence of the PCR product showed 94% nucleotide identity with the corresponding fragment in the Methylomonas LW 15 mmoX gene. Lake Constance Isolate 2 (Strain LC 2) Strain LC 2, a type I methanotroph, was isolated from a gradient enrichment culture obtained from an 8 × 10-3 dilution of the sediment. The pale-pink colonies were slimy with an aqueous consistency. They maintained their consistency after repeated streaking on mineral medium with agarose, but their size was reduced after successive transfers. Cells were actively motile, large, coccoid to oval in shape and sometimes changed to elliptical or rod-like shape, 1 - 2 x 2 - 3.5 µm in size (Figs 4a, b). They were surrounded by capsular material, were fragile and bursted easily upon applying little force to the cover slip. On solid mineral medium, mucoid colonies grew within 2 - 3 weeks at 16 20°C. Old plates often smelled of formaldehyde, and the presence of formaldehyde in the gas phase was confirmed by GC analysis of unopened desiccators in which this bacterium was growing (0.3 – 0.7%). When grown in closed glass vessels, formaldehyde was produced in the range of 0.06% - 1.4% in the gas phase, corresponding to 0.05 to 1.2 mM in the culture liquid. In liquid culture, strain LC 2 grew as a thin biofilm attached to the bottom of the glass tube. If the tube was vortexed the biofilm broke up into threads or fragments. Liquid cultures consisted mainly of chains or groups of cells.

40

Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen In gradient cultures, strain LC 2 usually formed a band at 20 - 22 mm distance from the air-exposed end. Occasionally an early lower band appeared after 10 - 12 days, and thus two bands were seen after 20 days of incubation at 16°C (Fig.4c). At the position of the band at 20 mm, methane and oxygen disappeared and oxygen was present only above the band (Fig. 1b). Both bands were sliced and observed by microscopy. They contained cell aggregates and the cells were much bigger compared to those grown on solid or in liquid medium of similar age. The average size of cells grown in gradients was 2.5 µm in diameter, whereas in liquid or on solid medium the average cell diameter was 1.5 µm. Whereas cells grown in liquid or on solid medium produced ample amounts of extracellular polymeric substances staining with alcian blue, cells grown in gradients carried very little of such extracellular polymers (not shown).

41


A nearly full-length (around 1400 bp) 16Sr DNA sequence was obtained by direct sequencing of the 27f-1492r PCR product with 27f, 1492r and MethT1df primers. To obtain the complete sequence of 1492 bp, the 16S rRNA partial gene was cloned and sequenced. All methods of tree construction applied yielded trees of similar topology. Strain LC 2 was distantly related to Methylobacter sp. (Fig. 2)). The closest cultured relatives of this strain are psychrophilic methanotrophs, Methylobacter psychrophilus (Tourova et al., 1999) and Methylobacter tundripaludum SV 96 species (Wartiainen et al., 2005), with 92.7 % and 93.4 % similarity, respectively (Fig. 2). The strain also contained a pmoA gene which could be amplified by both primer pairs (A189f-A682r, A189f-mb661r). The partial pmoA sequence of 530 bp was at the nucleotide level only 86% identical with that of cultured Methylobacter species, Methylobacter sp.BB5.1 and Methylobacter sp. LW12, and did not have an Msp I enzyme restriction site. It showed a distinct position in the phylogenetic tree close to the Methylobacter pmoA sequences (Fig. 3).

42


Isolated type II methanotrophs Also two type II methanotrophs were isolated from the gradient cultures enriched from two different sediment samples. Strains LC 3 and LC 4 were assigned to strains of Methylosinus sporium, by comparison of the sequence of the partial pmoA gene. Strains LC 3 and LC 4 had identical pmoA sequences which are very similar to Methylosinus sporium strain RG, a strain isolated previously from Lake Constance (Bussmann et al., 2004) and M.sporium SE 2. These strains were not characterized any further.

43


Table 1 pmoA clone libraries obtained from gradient culture bands Gradient Sampling Dilution culture date and bands incubation temperature

a

Approx. distance from air exposed end (mm) 5

Ab

May 2004, 20°C

3 x 10-4 and 1 x 10-5

Bb

Sept 2004, 20°C

8 x 10-3

3

Cb

July 2004, 20°C

8 x 10-3

45

Relative abundance of RFLP group

Nucleotide similaritya

88.6%, gradient clone A1 5.8%, gradient clone A2 100%, gradient clone B1, 100%, gradient clone C1,

95-97% Methylomonas sp. dito

95-97% Methylomonas sp.

92% Methylobacter SV 96

by NCBI blast b primers A189f-mb661r were used for all clone libraries

44


Table 2 Relative abundance of methanotrophic groups based on frequencies of pmoA genes in clone libraries Phylogenetic Clone group group Type I

Type II

No. of clones

Relative abundance (%)

B1a

42

66

Clones B67, A55, clone 63 (Pester et al., 2004) (91-99%)

B2a

2

3

Clones B9, A81, B39 (Pester et al., 2004) (9197%)

B6a

1

2

Clones B40, B77 (Pester et al., 2004) (92-97%)

B8b

2

3

pLWPmoA-2 (Nercessian et al.) (92%)

b

B9

3

5

PLWPmoA-11 (Nercessian et al.) (84%), Methylobacter BB5.1 (84%), Methylomonas sp. LW15 (83%)

B10b

8

13

W9_661_12, clone 9_661_3 (Erwin et al., 2005) (86%)

B7a

6

9

Clone 76 (Bussmann et al., 2004) (99%)

a

as identified in (Pester et al., 2004)

b

new groups identified in this study

Next relative (sequence identity)

Bacterial diversity in the growth bands To estimate the methanotrophic diversity in the growth bands three pmoA clone libraries were constructed (Table 1). Clone libraries constructed from bands A and B were dominated by sequences similar to pmoA sequences of strain LC 1 or of Methylomonas sp. LW 15 (Fig. 3). These gradient clones (A1, A2 and B1) were close to sediment clone group B9. Bands A and B were formed at 5 and 3 mm from the airexposed end of the tube. The third clone library was prepared from band C which was 45 mm from the air-exposed end. This library was dominated by a single group (gradient clone C1) which was within the most dominant sediment clone group B1 (Fig. 3). To estimate the overall bacterial diversity in these growth bands 16S rRNA gene clone libraries were constructed from DNA of the bands B and C. In band B a 45

Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen high bacterial diversity was found. Methanotrophic bacteria represented only 20% of the clone library, (99% similar to strain LC 1 and Methylomonas sp.LW15). The clone library was dominated by Flavobacteria (48% of the clone library) and betaproteobacteria similar to Pseudomonas saccharophila or Matsuebacter spp (17% of the clone library). In contrast, 16S rRNA gene clone library from band C showed less bacterial diversity. Here, a methanotroph dominated the clone library with 80% and the sequence was 98% close to Methylobacter tundripaludum SV96 by blast search. The remaining 20% were represented by beta-proteobacteria (similar to Pseudomonas saccharophila or Matsuebacter spp). Methanotrophic diversity in the sediment pmoA clone libraries were constructed using DNA from littoral sediment of Lake Constance. These libraries were prepared using the A189f - mb661r primers. The sequences obtained in this library were compared to sequences obtained in an earlier study (Pester et al., 2004), which were done using A189f - 682r primers. A total of 64 clones with the correct insert size were divided into eight clone groups after phylogenetic analysis (Fig. 3). Most of the clone groups were the same as identified before (Pester et al., 2004), and are named in the same way (Table 2). These groups were B1, B2, B6 (type I MOB) and B7 (type II MOB); whereas some new groups B8, B9 and B10 (type I MOB) were identified in the present study. Clone group B1 was the most dominant clone group with 65.5 % of relative abundance. Clone group B8 was closely related to clones from Mono Lake California (Lin et al., 2005) and Lake Washington (Nercessian et al.). Clone group B9 was only distantly related to known methanotrophs (Table 2) but grouped close to the branch where Methylomonas sp. LW 15, strain LC1 pmoA and gradient clones A1, A2 and B1 were

46

Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen present. Clones from group B 10 were not closely related to any cultured methanotroph but related to clones from a river plain aquifer (Erwin et al., 2005). Discussion Growth of MOB in gradients Based on a previous study (Amaral & Knowles, 1995), we describe here a cultivation system for methanotrophic bacteria that allows the incubation of numerous culture tubes in opposing gradients of methane and oxygen, thus mimicking life conditions of methanotrophs in sediments. Methanotrophs grew as bands instead of single colonies in the semi-solid agarose medium. Two types of bands of methanotrophic bacteria occurred, one growing approx. 5 mm below the air-exposed end and a lower one approx. 20 mm or 40 mm from the air-exposed end. The upper bands consisted of type I and type II MOB, but were usually dominated by type I MOB related to Methylomonas spp. as revealed by pmoA clone library analysis (Table 1), 16S rRNA gene library studies and by fluorescence in situ hybridisation studies (M.Rahalkar, unpublished data). Amaral et al. (Amaral & Knowles, 1995) only describe type I MOB at this position. Also, the known methanotrophs Methylosinus trichosporium (type II) and Methylobacter luteus (type I) both grew at the upper position, as did strains LC 3 and LC 4 (Methylosinus sporium strains, type II). The lower band, approx. 20 mm or 40 mm from the air-exposed end, consisted only of type I MOB related to the Methylobacter group (Table 1). Amaral et al. (Amaral & Knowles, 1995) described rare occurrence of such a lower band that was dominated by type II methanotrophs. This discrepancy may be due to the use of different inocula (freshwater sediment versus swamp and humisol samples) and different gas concentrations applied to the gradients.

47

Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen Table 3 Position of and flux of oxygen and methane to methanotrophic bands growing in gradient tubes. Culture

Approx.

Oxygen flux* -1

-2

Methane flux* -1

Flux ratio

-2

position of the (nmol h cm ) (nmol h cm ) (O2 / CH4) band (mm) Strain LC 1a

5

8.6

2.8

3.0

Sediment enrichmentb

5

7.9

1.7

4.8

strain LC 2a

20

9.5

4.3

2.2

22

8.1

3.9

2.1

Sediment enrichment

b

* Fluxes were calculated according to Fick’s first law of diffusion (F = -Ø x Ds x dc/dz), with the porosity Ø ≈ 1, diffusion coefficient DsO2 or CH4 = 2.3 or 1.9 x 10-5 cm2 s-1, change of oxygen or methane concentration versus depth (dc/dz). a see Fig. 1. b data not shown The methane flux was higher to the lower bands with only type I MOB than to the upper bands with type I and II, while the oxygen flux to both bands was similar (Table 3). It is assumed that type II MOB are dominant at high methane concentrations, and that type I MOB dominate at rapidly changing growth conditions (Graham et al., 1993; Macalady et al., 2002). However, a more detailed study on rice soil showed that there is no clear prevalence of type I or type II populations at different regimes of methane concentration (Henckel et al., 2000). Type I MOB appear to be more flexible, and were present at both positions in our study. The oxygen-to-methane flux ratio was 2.1 and 2.2 in the bands at the lower position in gradient cultures of strain LC 2 and of a sediment enrichment (Table 3). This is close to the theoretical ratio of two, suggesting that methane was completely oxidized to carbon dioxide. However, for most methanotrophic cultures and also for gradient enrichment cultures ratios of 1.6 – 1.8 are reported (Amaral & Knowles, 1995), 48

Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen reflecting the fact that substantial amounts of methane are assimilated after incomplete oxidation (Joergensen & Degn, 1983). The upper bands, observed in sediment enrichments exhibited higher oxygen-to-methane ratios which might be due to utilization of hydrolysed agarose constituents or exudates of the MOB. The presence of oxygen consuming, heterotrophic bacteria in the upper bands is also supported by the higher bacterial diversity in the upper bands, as shown by the 16S rRNA gene clone library. For growth in gradients it is essential that bacteria are motile to reach their optimal position in the gradient. Motility also allows the bacteria to adjust their position whenever the substrate supply changes. Hence, all our isolated strains are motile, as observed microscopically and as this is known for most strains of MOB (Hanson et al., 1991). This may be also the reason why we did not isolate any Methylocystis (type II) or Methylococcus (type I) strains, which are non-motile, were isolated (Bowman, 2000). Unfortunately, at least after the 4th transfer from gradient tube to gradient tube, bacteria stopped growing. We checked for reasons (medium composition, inoculum size, transfer procedure), but so far we could not solve this problem. Thus, we used the gradient system only for an initial enrichment step, and switched for isolation to a “conventional” cultivation procedure. Nonetheless, all isolated strains could grow again in gradients, as did also some tested strains from culture collections. The oxidation of methane to CO2 proceeds through methanol and formaldehyde which may be bound to various carriers for further oxidation to a formyl derivative (Vorholt, 2002). The equilibrium of methanol oxidation with pyrroloquinoline quinone or cytochrome c as electron acceptor is far on the side of formaldehyde. Thus, it is not surprising that formaldehyde is accumulated by methane-oxidizing

49

Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen bacteria under excess supply with methane and oxygen, and this was found also with our strain LC 2 during growth on solid medium plates. So far, formaldehyde production (0.2 - 7 mM) by methanotrophs was reported only during grow with high methanol and oxygen concentrations (Agrawal & Lim, 1984; Costa et al., 2001). Formaldehyde becomes inhibitory at concentrations between 1 mM (Hou et al., 1978) and 7 mM (Costa et al., 2001). Thus, the amount of formaldehyde produced by strain LC 2 (up to 1 mM) could be sufficient to inhibit growth of this bacterium, and other strains might be even more sensitive to this toxic compound. Methanotrophs are known to produce copious amounts of exopolymeric substances (EPS) (Hou et al., 1978; Linton et al., 1986). For mixed MOB cultures in compost, the highest EPS production was reported at high oxygen levels (10.5% versus 1.5% O2) (Wilshusen et al., 2004). EPS can act as a micro-scale diffusion barrier since the apparent diffusion coefficient can be 50 times smaller than that of an aqueous solution (Guiot et al., 2002). In landfill cover soil EPS impeded oxygen diffusion to an active biofilm and limited the extent of methane oxidation (Hilger et al., 2000) and EPS formation has been observed also as a response to stress caused by exposure to toxic substances such as detergents (Schleheck et al., 2004); Klebensberger et al., personal communication]. On the other hand, EPS production can impair growth because of the energy cost involved (Kreft & Wimpenny, 2001). Our strain LC 2 produced EPS mainly when grown on solid or in liquid medium, perhaps as a means to protect the cells from oxygen stress, and only little EPS was formed during growth in gradient tubes. The absence of formaldehyde and EPS formation along with increased cell size in gradient cultures of strain LC 2 indicate that under these conditions the diffusion-limited access of methane and oxygen avoids oxygen stress and helps to prevent self-intoxication.

50

Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen Isolation of methanotrophic bacteria Gradient cultivation was described first by Knowles et al. as a novel enrichment culture technique for the growth of methanotrophs (Amaral & Knowles, 1995). Only one isolate Methylobacter sp. T 20 obtained by such enrichment has been described (Ren et al., 2000). Our study demonstrates a further strategy which could be used for isolation of methanotrophic bacteria from aquatic habitats. As methanotrophs were associated with other bacteria, it was difficult to isolate them directly in gradient cultures. Thus, we used initial enrichment in gradients followed by subsequent transfers into liquid or on solid medium. Strain LC 1 is phylogenetically closely related to Methylomonas methanica and Methylomonas sp. LW 15, which both were isolated from Lake Washington (Auman et al., 2000), and Methylomonas sp. LW 15 is one of those from this site which possesses sMMO. This enzyme has been found in few type I MOB, e.g., Methylomonas strains from oil- or TCE-contaminated sites, as well as from Lake Washington, i.e. a freshwater habitat (Auman & Lidstrom, 2002). Strain LC 1 possesses sMMO, as confirmed by PCR amplification of the partial mmoX gene. The mmoX gene, which encodes the α subunit of the hydroxylase component of sMMO, has been used as a biomarker for sMMO (Auman et al., 2000; McDonald et al., 1995; Shigematsu et al., 1999). In addition, strain LC 1 has also the exact match for rmonas3X probe (860-879 bp) which was an oligonucleotide probe designed specifically for sMMO-containing Methylomonas strains in a study on Lake Washington (Auman & Lidstrom, 2002). Hybridisation studies suggested that Lake Washington sediment is dominated by sMMO-containing Methylomonas-like type I methanotrophs (Auman & Lidstrom, 2002). Thus, we isolated a sMMO-containing

51

Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen type I MOB, which had not yet been described for Lake Constance yet but might be a dominating MOB here as well. Strain LC 2 represents a novel lineage in the phylogenetic tree of type I MOB, and is present on a branch close to that of psychrophilic MOB, like Methylobacter psychrophilus or the newly described Methylobacter tundripaludum SV96 (Wartiainen et al., 2005) (Fig. 2). This phylogenetic branch also contains Methylobacter sp. T20, which is the only methanotroph isolated from a gradient culture before (Ren et al., 2000). Other members present on this branch are mostly methanotrophic clones from various stable-isotope-probing experiments. A 16S rRNA gene similarity of 92-93% of strain LC 2 with known species indicates its novelty. The pmoA sequence is also novel and more related (89%) to pmoA clones MLA-49, MLA-39 etc. which were isolated from Mono Lake, California (Lin et al., 2005), clones B67, clone 63 and A55 from Lake Constance (Pester et al., 2004), and only 8386 % related to cultivated Methylobacter species. The amino acid similarities are: 91% similar to Methylobacter sp. LW 12 and Methylomicrobium buryatense, and 93% similar to clone B67 and clone A55 from Lake Constance littoral sediment. Morphologically similar bacteria have been observed and enriched from Russian arctic Tundra regions (Berestovskaya et al., 2002). These have been described as morphotype 2, which are cocci of 2 - 2.5 µm diameter, with mucous capsules, preferred growth temperatures of 5 - 10° C, and pH of 5 - 7. Diversity and abundance of MOB in sediment compared to gradient cultivation The methanotrophic diversity within the bands was low as judged by the pmoA clone libraries (Table 1). However, within a particular growth band, the growing community represented a differential bacterial diversity (e.g. high diversity in case of

52

Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen band B and low diversity in case of band C) as revealed by 16S rRNA gene clone libraries. The results of gradient pmoA clone libraries were compared with the pmoA clone library of Lake Constance littoral sediment prepared in these studies. Thus for comparison, sediment pmoA clone libraries had to be prepared using A189f-mb661r primers because these primers are more specific in amplifying only the pmoA gene and not the amoA (Costello & Lidstrom, 1999) as well as are known to cover more diversity of MOB (Bourne et al., 2001). The relative abundance of methanotrophs based on frequencies of pmoA genes in the sediment clone library shows a dominance of type I methanotrophs (91%). Within the type I MOB, clone group B1 or Methylobacter like MOB dominate with 65%. In our gradient cultures, gradient clone group C1 lies within this dominant group, and we also isolated the methanotrophic strain LC 2, which is close to this group but has a distinct phylogenetic position. Another interesting group which we could cultivate was the group of the gradient clones A1, A2 and B1 together with strain LC 1, distantly related to sediment clone group B9. Among the type II MOB, the clone group B7 is dominant, and isolates of this group have been obtained by gradient (strains LC 3 and LC 4, this study) and non-gradient cultivation (Bussmann et al., 2004). Thus, gradient cultivation appears to broaden the diversity of cultivable methanotrophs substantially. So far, no MOB from other clone groups related to Methylococcus (clone groups B2 and B6) or clone groups related to uncultured methanotrophs (B8 and B10) which represent a total of 20% sediment clone library have been cultivated from Lake Constance sediment. Further studies including gradient cultivation will aim at isolation of representatives of this group as well. In this context, strategies have to be developed to allow single colonies to grow in gradients which has not been achieved successfully so far. Thus,

53

Chapter 2: Methanotrophic bacteria in opposing gradients of methane and oxygen our present study is a further step in using gradient cultivation as a tool to cultivate novel methanotrophs by mimicking the natural conditions in aquatic systems. Acknowledgements This study was supported by the Deutsche Forschungsgemeinschaft (SFB 454) and research funds of the Universität Konstanz. We wish to thank Thomas Grützner for assistance in the formaldehyde determination, Dirk Schmitt-Wagner and Ulrich Stingl for introduction to the ARB software package, and Michael Pester for valuable discussions.

54

Chapter 3: A novel methanotroph isolated by gradient cultivation

Chapter 3 Characterization of a novel methanotroph, Methylosoma difficile gen. nov., sp. nov., enriched by gradient cultivation from littoral sediment of Lake Constance Monali Rahalkar, Ingeborg Bussmann and Bernhard Schink * Submitted to International Journal of Systematic and Evolutionary Microbiology (In revision)

Summary A novel methanotroph, strain LC 2T, was isolated from the littoral sediment of Lake Constance by enrichment in opposing gradients of methane and oxygen, followed by traditional isolation methods. Strain LC 2T grows on methane or methanol as its sole carbon and energy source. It is a Gram-negative, non-motile pale pink-colored methanotroph showing typical intracytoplasmic membranes arranged in stacks. Cells are coccoid, elliptical or rod-shaped and occur often in pairs. Strain LC 2T grows at low oxygen concentrations and in counter gradients of methane and oxygen. It can grow on medium free of bound nitrogen - , possesses the nif H gene and fixes atmospheric nitrogen at low oxygen pressure. It grows at neutral pH and at temperatures between 10 and 30 oC. Phylogenetically it is most closely related to the genus Methylobacter, with M. tundripaludum and Methylobacter psychrophilus related by 94% and 93.4%, respectively. Also the pmoA gene of strain LC 2T is most closely related to pmoA genes of Methylobacter, (92% similar to Methylobacter sp. LW 12 by derived amino acid sequence identity). The DNA (G+C content) is 49.9 mol% and the major cellular fatty acid is 16:1ω7c (60%). Strain LC 2T is described as type strain of a new genus and species, Methylosoma difficile (=JCM 14076T=DSMZ#### ) 55

Chapter 3: A novel methanotroph isolated by gradient cultivation Key words: Gradient cultivation, pmoA, Lake Constance, Methylosoma difficile Subject Category: New Taxa (Proteobacteria)

56

Chapter 3: A novel methanotroph isolated by gradient cultivation Introduction Aerobic methane-oxidizing bacteria (MOB) or methanotrophs are a unique and important group of bacteria which act as natural filters controlling the release of methane, an important greenhouse gas, from anoxic sediments and soils. Aerobic MOB in freshwater environments such as lake sediments are active at the zone where methane and oxygen meet. In a previous study from our lab, a specific cultivation setup was developed in which methanotrophic bacteria were grown in opposing gradients of methane and oxygen (Bussmann et al., 2006). A novel bacterial strain, LC 2T, was isolated which was found to be moderately related to the MethylobacterMethylosarcina group (92 - 94% similarity) and less related to the other genera of MOB (similarity 90 - 91%) e.g. Methylomonas, Methylomicrobium, as determined by 16S rRNA sequence comparison. The closest relatives of this new strain were Methylobacter sp. such as Methylobacter psychrophilus (Tourova et al., 1999) and the newly described Methylobacter tundripaludum strain SV 96 (Wartiainen et al., 2006), which are either psychrophilic or have been isolated from cold habitats, respectively. In culture-independent studies, we found that littoral sediment of Lake Constance is dominated by Type I MOB (Bussmann et al., 2006; Pester et al., 2004). Among those Methylobacter- like MOB (clone group B1) dominated clone libraries of partial pmoA gene (Bussmann et al., 2006) amplified with A189f-A682r primers (Holmes et al., 1995) as well as with A189f-mb661r primers (Bussmann et al., 2006; Costello & Lidstrom, 1999) and contributed to the major peaks observed in T-RFLP studies from the littoral sediment of Lake Constance (Pester et al., 2004). Recently, many pmoA and 16S rRNA gene sequences related to the Methylobacter genus have been reported as dominant groups in other freshwater habitats such as freshwater wetland marshes (Bodelier et al., 2005), lakes such as Mono Lake (Lin et al., 2005) and Lake

57

Chapter 3: A novel methanotroph isolated by gradient cultivation Washington (Nercessian et al., 2005), estuarine habitats (McDonald et al., 2005), as well as in chironomid larvae in lake sediments (Eller et al., 2005). Until now, a total of thirteen genera of methanotrophs have been described, which belong to either the classes Alphaproteobacteria (Type II MOB) or Gammaproteobacteria (Type I MOB). Recently it was discovered that Crenothrix polyspora (Cohn) (Stoecker et al., 2006), a well-known; uncultured filamentous bacterium, is a methane oxidizer, and its 16S rRNA genes (total 4 clusters) formed a new group within the Methylococcaceae family which also branched near Methylobacter psychrophilus and related methanotrophs. The purpose of the present study is to formally characterize strain LC 2T and to determine its correct taxonomical position. Although this strain was isolated from a gradient culture it could grow on solid and in liquid media as well. Methods Isolation and growth conditions Strain LC 2T was isolated from littoral sediment of Lake Constance after an initial enrichment in a gradient culture followed by transfer into liquid dilution series and finally on solid agarose medium, as described in detail (Bussmann et al., 2006). Cells were grown in dilute mineral medium (Bussmann et al., 2006) either in liquid medium, on solid medium (with 1.2 % agarose NEEO, Ultra quality, Roth, Karlsruhe, Germany) with 0.01 % cycloheximide, or in a gradient system (0.2% agarose) with opposing gradients of methane and oxygen as described before (Bussmann et al., 2006). Growth on 1.5 % agar (BD Biosciences) was also checked. The diluted medium as described in (Bussmann et al., 2004) contained the following salts (per liter) : 0.1 g NaCl, 0.04 g MgCl2 x 6 H2O, 0.05 g KCl, 0.015 g CaCl2 x 2 H2O, 0.016 g Na2SO4 and trace element solution SL 10 (1ml l-1) (Widdel, 1988). K-Na-phosphate

58

Chapter 3: A novel methanotroph isolated by gradient cultivation buffer (pH 7.2) and KNO3 were added to final concentrations of 150 µM and 50 µM, respectively, and the medium was buffered to pH 7.2 with 0.01 M HEPES buffer. In addition, a seven-vitamin solution (Widdel & Pfennig, 1981) was added (1ml l-1). Strain LC 2T was checked also for growth in: a) Classical nitrate mineral salt medium (Whittenbury et al., 1970) with the trace element solution replaced by SL 10 solution, b) Low nitrate mineral salt medium (1/10th concentration of potassium nitrate, pH 7.0 with HEPES buffer) and c) Medium with 10-fold concentrated version of the above mentioned mineral medium (Bussmann et al., 2004). The effect of copper on growth was checked by inoculating the culture with additional 1 - 5 µM CuSO4 in liquid medium. The basal concentration of Cu in the mineral medium was very low, i.e. 0.011 µM. Strain LC 2T was grown in 100 ml flasks or 15 ml glass tubes with 25 – 30 ml or 5 ml medium, respectively, in closed desiccators. The strain was maintained by streaking single colonies on solid agarose plates, or by inoculating a single colony in liquid medium and then streaking the grown liquid culture on agarose plates. Until about one year after isolation, strain LC 2T was grown in closed desiccators under a gas atmosphere of 24% CH4, 2% CO2, 17% O2, and the balance N2 (Bussmann et al., 2006). As the strain grew well in gradients and appeared to grow at low oxygen tension, (Bussmann et al., 2006) the gas atmosphere for growing this culture was slightly modified to 20 % methane, 2 % CO2, 30 % air (around 6 % O2) and the balance N2. All growth experiments performed in this study were performed with this modified gas atmosphere in the dark at 16 ºC or 21 ºC, unless otherwise mentioned. The strain was also tested for growth in closed 150 ml bottles with different volumes of liquid medium and a gas phase of methane and air (20:80). Possible presence of heterotrophic contaminants was checked every time by plating on 1:10 diluted nutrient agar plates with additional 0.05% yeast extract. For long-term storage,

59

Chapter 3: A novel methanotroph isolated by gradient cultivation glycerol stocks were prepared and preserved at -70oC. Survival of the strain after such preservations was checked by inoculation into fresh medium. Cultures were also preserved at 4oC for long term storage. Ten % inoculum was used in all growth experiments and the incubation period was eight weeks. Since the bacteria tended to grow as a faint pink biofilm at the bottom, the tube or flask was shaken well to suspend the cells uniformly before the OD570nm was recorded. Growth was always confirmed by phase contrast microscopy. Morphological characterization and electron microscopy Cells were observed under a phase contrast microscope (Axiophot; Zeiss) and photographed with a cooled charge-couple device camera (Magnafire, INTAS). For fixation for electron microscopy 50 ml of an exponentially growing culture was spun down at 5000 g. The cell pellet was washed once with PBS (50 mM potassium phosphate buffer pH 7.5 plus 0.9% NaCl) and then suspended in 1ml of PBS. 140 µl of 25% glutaraldehyde was added to the cell suspension and then incubated overnight at 4oC. The cells were centrifuged again at 5000 g. The pellet was washed again in PBS and finally suspended in around 150 µl of PBS. After chemical fixation, cells were embedded in 1.5% (w/v) molten agar (final concentration). The agar block was cut into small pieces of 1 mm3 volume and the pieces were dehydrated in a graded methanol (v/v) series (15%, 30% for 15 min; 50%, 75%, 95% for 30 min; 100% for 1 h) under concomitant temperature reduction to -40 °C. The samples were infiltrated with Lowicryl K4M resin (in methanol 50%, v/v, for 1 h; 66%, v/v, for 2 h;100 % for 10 h), then polymerized for 24 h at -40 °C and for three days at room temperature (Roth et al., 1981; Hoppert and Holzenburg, 1998). Resin sections of 80-100 nm thickness were cut with glass knives. Electron microscopy was performed in a Philips EM 301 transmission electron microscope at 80 kV with calibrated magnifications. To

60

Chapter 3: A novel methanotroph isolated by gradient cultivation check for the presence of cellular appendages like flagella or pili, negative stainings were prepared with fresh cell material. Utilizable carbon and nitrogen sources Utilization of various carbon sources was studied in liquid mineral media supplemented with either one of the following filter-sterilized substrates (0.1% w/v): formate, formamide, arabinose, raffinose, lactose, maltose, xylose, glucose, fructose, and sucrose. The ability of the strain to grow on methanol and formaldehyde was tested at lower concentrations, i. e. 10 mM to 50 mM methanol and 1 mM to 50 mM formaldehyde. Other substrates like acetate (2 mM and 10 mM) were tested for growth in liquid medium to check for heterotrophic growth (Dedysh et al., 2005) of strain LC 2T. Nitrogen sources were tested with liquid medium in which KNO3 was replaced by one of the following compounds at 0.05 % (w/v): NH4Cl, urea, glycine, serine, valine, asparagine, aspartate, L-glutamic acid, glutamate, peptone and yeast extract. To test the ability of the culture to fix atmospheric N2, media free of bound nitrogen compounds were used. The acetylene reduction test was done with cultures grown with 10 mM methanol without any bound nitrogen source, as modified by (Auman et al., 2001). Briefly, 25 ml of such grown culture was transferred to a bottle with a rubber stopper and was gassed with nitrogen; air and acetylene were added (89% N2: 9% air: 1% acetylene) and incubated overnight. The gas phase was checked for ethylene production by gas chromatography. Optimum pH, temperature and salt content The optimum pH and temperature ranges were determined in liquid medium. Growth at pH values in the range of 2.6 to 9.0 was checked after buffering the medium with citrate-phosphate buffer (pH 2.6 to 6.6), HEPES buffer (pH 7 and 7.5) and glycine buffer (pH 8 and 9). Growth was also checked without using any buffer,

61

Chapter 3: A novel methanotroph isolated by gradient cultivation but using only HCl or NaOH for adjusting the pH. Strain LC 2T was grown at a temperature range of 4 oC to 37 oC, in liquid media. To test the optimum salt concentration, additional NaCl (0.5%, 1%, 1.5% and 2.0%, w/v) was added to the mineral medium. The basal NaCl concentration in the mineral medium was 0.01% (w/v). Resistance to desiccation and heat Heat resistance was tested by heating cell suspensions at 50, 60, 70 or 80oC for 10 minutes each followed by plating onto solid medium, and incubating at optimal conditions for two to three weeks. Desiccation resistance was assessed according to Whittenbury et al. (1970) by air drying suspensions of strain LC 2T on glass slides and then inoculating into medium after an interval of 1 week up to four weeks. Formation of exospores was checked for by heating a 3 - 4 weeks old culture at 80oC for 20 minutes and then looking for colony formation after incubation under standard conditions (Bowman et al., 1993). Cysts were stained according to (Vela & Wyss, 1963). Cellular fatty acid analysis Phospholipid fatty acid analyses were performed at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Braunschweig, Germany. Strain LC 2T was grown in 200 ml flasks, in closed desiccators. Cells were pelleted, freezedried and were send for PLFA analysis. There, the cells were saponified, methylated and the methyl esters were extracted and subjected to gas chromatography. The gaschromatographic elution profile of the fatty acid methyl esters was compared with the fatty acid patterns stored in the fatty acid database of the Microbial Identification System (MIS, MIDI Del. USA) and qualitative and quantitative composition of the pattern were given.

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Chapter 3: A novel methanotroph isolated by gradient cultivation DNA extraction, phylogenetic analysis and G+C content DNA was extracted and the complete 16S rRNA gene sequence was determined as previously described (Bussmann et al., 2006). 16S rRNA gene sequences of Type I methanotrophs of the family Methylococcaceae along with newly described sequences of Crenothrix polyspora and sequences of some clones were obtained after blast search. Phylogenetic analysis was done using the ARB software package (version 2.5b; http://www.arb-home.de) (Ludwig et al., 2004). The new sequences were added to the ARB database and aligned using the FAST Aligner tool as implemented in ARB. Alignments were checked and manually corrected where necessary. Sequences of 1419 nucleotides were used for alignment. Only those positions which were identical in 50 % of all sequences were used to create a filter. Phylogenetic analysis was done using the maximum likelihood, neighbour-joining and maximum parsimony algorithms as implemented in ARB (Ludwig et al., 2004). Phylogenetic distances were also determined by using the similarity matrix in ARB without using any filter and also with E. coli as the filter. Phylogenetic analysis of the pmoA gene was done as described earlier, and phylogenetic trees were constructed based on 164 amino acids (Bussmann et al., 2006). Mol% G+C content was measured by HPLC adapted from (Tamaoka & Komagata, 1984) in DSMZ and calculated according to the method of (Mesbah et al., 1989). Presence of sMMO and nitrogenase The presence of sMMO was checked by PCR amplification of the mmoX gene with primer pairs mmoXA - mmoXB (Auman et al., 2000) as well as by colorimetric assay (Graham et al., 1992). To check for the presence of nitrogenase, the nif H gene was amplified as described (Poly et al., 2001), partially sequenced and subjected to blast search at the NCBI (http://www.ncbi.nlm.nih.gov/) (Altschul et al., 1990).

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Chapter 3: A novel methanotroph isolated by gradient cultivation Results and Discussion Isolation and growth characters of strain LC 2T Strain LC 2T was isolated after enrichment in a gradient culture system obtained after a final dilution of 8 x 10-4 of the littoral sediment, after further transfers on liquid and solid media (Bussmann et al., 2006). The strain was isolated from a mixed culture in which it was associated with thin rods. When isolated, the colonies were much bigger, and more mucoid and watery than after extended cultivation. This could be due to a tendency of the cells to form aggregates or to the presence of an initially contaminating bacterium. The strain grew in liquid culture at low oxygen tensions up to 10 % air, i.e. 2 % oxygen. In gradients, it formed 2 bands, the deepest one being 32 mm away from the air-exposed end (Bussmann et al., 2006). Strain LC 2T also grew well on 1.5 % agar although routinely agarose was used. The strain could not be maintained on solid medium by repeated transfers. Therefore, always a single colony was grown in liquid culture and then streaked on solid medium. No active motility could be detected 2 years after isolation. Cells occurred singly or in pairs or sometimes in chains of 4 - 5 cells (especially on solid medium) or in aggregates. In liquid medium, strain LC 2T formed a mucoid pink biofilm at the bottom of the flask. If the flask was shaken, a whorl of mucoid biofilm appeared which was held together at a small point in the centre of the flask. After shaking, the cells could be suspended uniformly. Cells also became oval or elliptical in shape and changed from coccoid to rod-shaped forms in older cultures. Liquid cultures grew better in magnetically stirred flasks inside a desiccator. Strain LC 2T resembled Methylobacter tundripaludum strain SV 96 in terms of color of the colony, and in cell size (Table 1). Comparison of strain LC 2T with other methanotrophic genera is shown (Table 1). Strain LC 2T did not survive in glycerol stock cultures. After initial growth at optimum temperature,

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Chapter 3: A novel methanotroph isolated by gradient cultivation the strain survived well at 4oC for 2 – 3 months. Poor growth was observed at increased Cu concentration. The strain grew well in dilute nitrate mineral salt medium and undiluted mineral medium but grew poorly in the classical nitrate mineral salt medium. It also grew well in non-shaken closed bottles (150 ml), with 15 – 20 ml of medium and 20% methane in the gas phase. DNA extraction, phylogenetic analysis and G+C content Strain LC 2T formed pale-pink colonies of around 1 -2 mm diameter on agarose plates and 2 -3 mm diameter on agar, after incubation for 2 - 3 weeks (Table 1). Coccoid cells were observed (often in pairs) by phase contrast microscopy which were 1.5 – 2 µm in length and approximately 1 µm in diameter Fig. 1(a). Intracytoplasmic membranes were arranged in stacks, mainly in the cell periphery, which is a typical feature of Type I methanotrophs (Whittenbury et al., 1970) Fig. 1(b). Cells contained large PHA-granules and, very likely, also glycogen granules which stained dark. Ultrathin sections of the cell periphery exhibited a typical appearance of a Gram-negative cell envelope with two dark thin layers of the outer membrane, a peptidoglycan layer located in the periplasm, and a cytoplasmic membrane Fig. 1(c). Flagella were absent, but pili of approximately 5 nm in width and up to 2 µm in length could be detected infrequently.

65


Table 1. Comparison of Strain LC 2T with other methanotrophic genera

CharacteristicsMethylomonas

Methylomicrobium Methylosarcina Methylosphaera Methylobacter

Strain LC 2T

* Cell

rods

rods

Sarcina shaped, or cocci

Rods or cocci

cocci

Morphology Motility

+

Cyst

+

+

Cocci, elliptical, or rods

variable

-

variable

-

variable

-

+

+

-

-

+

+

+

White

Light brown-buff

Yellow, brown or

Pale pink

formation Nitrogenase variable genes Pigmentation Pink, yellow ochre

pale pink †

G+C content 51-59

50-60

53-54

43-46

(Mol %)

* - except Methylosarcina lacus †- Methylobacter tundripaludum

66

49-54

49.9


Utilizable carbon and nitrogen sources Strain LC 2T grew only on methane or on methanol (10 mM to 50 mM). No other carbon substrates were utilized. Of the different N sources checked, it utilized nitrate, L-glutamine, L-glutamic acid, L asparagine, and L-aspartic acid. Growth on organic N sources such as peptone and yeast extract was better and faster, as compared to nitrate. Strain LC 2T grew without any bound nitrogen source under standard gas conditions. The acetylene reduction test was positive although the ethylene peak was very small and the reaction required overnight incubation. Effect of pH, temperature and NaCl concentrations on growth Strain LC 2T grew in the pH range of 5 – 9 when no additional buffers were used, with the best growth in between pH 6 to 8. When the media were buffered, growth was only around the neutral pH, which might be due to the sensitivity of the strain towards high concentration of organic compounds. Strain LC 2T grew in a 67

Chapter 3: A novel methanotroph isolated by gradient cultivation temperature range of 16 ºC to 30 ºC, the optimum growth temperature being around 25 ºC. The specific growth rates at 16 ºC, 25 ºC and 30 ºC were 0.0024, 0.0041, and 0.0065 per h. Even though growth was fast at 30 ºC, the growth declined after reaching around 0.15 OD570nm, which might be due to imbalance of its metabolism. Little growth was observed at 10 ºC. No growth was observed at 37oC and 4oC. NaCl added to the medium to concentrations of 0.5 to 2 % inhibited growth. Resistance to desiccation and heat, and exospore formation Strain LC 2T grew after a heat shock at 50oC and 60oC for 10 minutes, but did not grow after a heat shock at 70oC. It formed neither micro colonies after exposure to 80oC, nor exospores. Even though cysts were frequently observed in older cultures, they were not resistant to desiccation for a week. Cellular fatty acid analysis Strain LC 2T showed a unique pattern of fatty acids compared to representatives of the related Type I methanotrophic genera Methylobacter, Methylosarcina, Methylomicrobium and Methylomonas, although the patterns of the Methylobacter genus were the closest (Table 2). Fatty acid patterns of Crenothrix polyspora were not available and thus could not be compared. The major fatty acid was 16:1ω7c. Strain LC 2T contained also 12:0 fatty acids which were observed only in the genus Methylosarcina. The percentage of 16:1ω7c, 14:0 and 16:0 were comparable to those of the neighbouring genus Methylobacter. Strain LC 2T had 15% of 16:1ω6c which was similar to the values found in the genera Methylomicrobium and Methylomonas. There was no 16:1ω5c or 5t fatty acid as seen in almost all other genera except Methylosarcina, which has a very low percentage of 16:1ω5c. In addition, strain LC 2T also showed minor amounts of 15:0, 16:1 ω11c and 16:0 3 OH fatty acids which were not found in other Type I methanotrophs so far. 68


Table 2.Cellular fatty acids (% of total) of strain LC 2T in comparison with other Type I methanotrophic genera Fatty acid Methylomonas* Methylomicrobium* Methylosarcina† Methylococcus* Methylobacter* Strain LC 2T

12:0

NR

NR

3±0.4

NR

NR

2.74

14:0

22±3

1±1

1.5±0.5