Methanotrophs and Methanogens in Masonry

Abstract

Methanotrophs were present in 48 of 225 stone samples which were removed from 19 historical buildings in Germany and Italy. The average cell number of methanotrophs was 20 CFU per g of stone, and their activities ranged between 11 and 42 pmol of CH₄ g of stone⁻¹ day⁻¹. Twelve strains of methane-oxidizing bacteria were isolated. They belonged to the type II methanotrophs of the genera Methylocystis, Methylosinus, and Methylobacterium. In masonry, growth substrates like methane or methanol are available in very low concentrations. To determine if methane could be produced by the stone at rates sufficient to support growth of methanotrophs, methane production by stone samples under nonoxic conditions was examined. Methane production of 0.07 to 215 nmol of CH₄ g of stone⁻¹ day⁻¹ was detected in 23 of 47 stone samples examined. This indicated the presence of the so-called “mini-methane”-producing bacteria and/or methanogenic archaea. Methanotrophs occurred in nearly all samples which showed methane production. This finding indicated that methanotrophs depend on biogenic methane production in or on stone surfaces of historical buildings.

Stone surfaces are an environment for microorganisms that shows wide variations of temperature and water content (31). Moreover, they are oligotrophic, and some show high salt concentrations (22). The low nutritional value selects for phototrophic, chemolithoautotrophic, and oligotrophic chemoorganotrophic bacteria which are among the primary flora of stones (4). Growth substrates for methanotrophs, like methane and methanol, are present in extremely low concentrations in masonry. They reach the stone surface as gaseous methane or, in the case of methanol, dissolved in rain and the water vapor of the atmosphere (7).

In most cases methanotrophs are found at the oxic-nonoxic interfaces of soils and sediments, where methane concentrations are high (12, 17, 19). Methane is produced mainly through the anaerobic decay of organic matter by methanogenic archaea. The so-called “mini-methane” producers, which belong to the proteolytic clostridia or the sulfate-reducing bacteria, can also produce small amounts of methane in side reactions of their metabolism (26, 27). It is not known whether methane production can occur on aerobic stone surfaces. Several reports have demonstrated the emission of methane from anaerobic microsites of aerobic biotopes like soils, lakes, and biofilters (21, 24, 32).

The aim of the experimental work described here was to isolate and characterize strains of methanotrophs from a habitat which up to now has not been investigated and, furthermore, to determine the cell numbers and activities of methanotrophs in samples from masonry. The biogenic methane production in stone samples was investigated because growth substrates for methanotrophs are present in very low concentrations (8).

MATERIALS AND METHODS

Stone material and inventory of cell numbers.

Stone samples from 19 historical buildings in Europe were analyzed to determine the possible presence of methanotrophic bacteria. All samples investigated were removed by hammer and chisel from a depth of 0 to 5 mm and then transferred to the lab, where they were ground to a grain size of 100 μm and stored up to 6 weeks at 4°C in the dark. CFU of methanotrophic bacteria were measured by the most probable number (MPN) technique with 10-fold dilutions of three replicates. A total of 5 g of ground stone material was suspended in 4.5 ml of nitrate mineral salts (NMS) medium (see below) in gastight 25-ml serum bottles sealed with rubber plugs. Before the MPN determination, the bottles were shaken for 5 min with a whirl-mix. The headspace of each bottle was filled with a gas mixture of 3% (vol/vol) CH₄ and 0.3% (vol/vol) CO₂ in artificially produced air (20% O₂, 80% N₂). The bottles were then shaken on a rotary mixer for 6 weeks at 28°C in the dark. The depletion of methane indicated the presence of methanotrophs. Methane was measured by gas chromatography as described previously (2). Stone material that had been autoclaved for 1 h at 115°C was used as a control.

Activity of methanotrophs in stone samples.

Fresh stone material with a water content of 3 to 8% (wt/wt) was used to determine the methane-oxidizing activity of methanotrophs. Therefore, 25-ml serum bottles were filled in each case with 10 g of ground stone material (100-μm grain size) and sealed gastight. The gas phase was adjusted to a methane concentration of 100 ppm (vol/vol) CH₄ at a pressure of 105 kPa. The consumption of methane was measured every 5 to 12 h for 1 week. Autoclaved stone material was used as a control. The water content of the stone samples was determined gravimetrically after the experiment.

Isolation of methanotrophs.

A modified NMS medium was used (35). The medium contained the following, per 1,000 ml of distilled water: NaNO₃, 850 mg; K₂SO₄, 170 mg; MgSO₄ · 7H₂O, 37 mg; and CaCl₂ · 6H₂O, 7 mg. After autoclaving, 10 ml of phosphate buffer solution, 0.5 ml of trace element solution, and 1 ml of iron solution were added.

The trace element solution (pH 4.0) contained the following, per liter of distilled water: ZnSO₄ · 7H₂O, 10 mg; MnCl₂ · 4H₂ · 4H₂O, 3 mg; H₃BO₄, 30 mg; Na₂MoO₄ · 2H₂O, 3 mg; CaCl₂ · 6H₂O, 20 mg; NiCl₂ · 6H₂O, 2 mg; and CuCl₂ · 2H₂O, 1 mg. The phosphate buffer contained 1.4 g of KH₂PO₄ and 3.6 g of Na₂HPO₄ in 100 ml of distilled water, pH 6.8. The iron solution contained 1.12 g of FeSO₄ · 7H₂O in 5 ml of 0.25 M H₂SO₄ in 100 ml of distilled water.

Methanotrophs were isolated in liquid culture as well as on agar plates. Therefore, enrichment cultures (3% [vol/vol] CH₄ atmosphere, NMS medium) from different stone samples were made and transferred several times. Cells from these cultures were diluted in NMS medium (10-fold dilution) with a 3% (vol/vol) methane atmosphere to obtain pure cultures of methanotrophs. Cells from enrichment cultures were also spread on agar plates. The plates contained NMS medium and 1% (wt/wt) Noble agar. After 2 weeks of incubation in 3% (vol/vol) CH₄, milky colonies were transferred into liquid medium. The absence of heterotrophic contaminants was tested by using complex agar (pH 7.4) containing the following, per liter of distilled water: meat extract, 0.5 g; Bacto Peptone, 0.5 g; yeast extract, 0.1 g; KH₂PO₄, 0.1 g; NaCl, 50 mg; and agar, 15 g. The uniform cell shape of pure cultures was examined by phase-contrast microscopy. The test organisms Methylomonas methanica (type I), Methylosinus trichosporium (type II), and Methylococcus capsulatus (type X) were able to grow under these isolation conditions.

Characterization of methanotrophs.

To determine the pathway of formaldehyde assimilation, cell extracts were tested for hexulosephosphate synthase activity (9) and hydroxypyruvate reductase activity (1). Formaldehyde was determined colorimetrically with the Hantzsch reaction (25). The protein concentration was measured by the method of Bradford (5). The growth of methanotrophs on 0.01, 0.1, and 1.0% methanol in sterile NMS medium was investigated.

The localization of intracytoplasmic membranes and the presence of capsule substances were identified by transmission electron microscopy. The cells were prepared according to the methods of Sabatini et al. (28) and Watson (33).

The ability of methanotrophs to oxidize naphthalene to naphthol, as a specific reaction of the soluble methane monooxygenase (sMMO), was determined as described by Koh et al. (20).

Methane production in stone material.

In order to measure methane production, 5 g of ground stone (100-μm grain size) was transferred into gastight 25-ml bottles and diluted in 4.5 ml of medium for either methanogenic archaea or mini-methane producers. As in the study of Whitman et al. (34) medium 1 with sodium acetate (50 mM) as an electron donor was used for methanogenic archaea. The antibiotics kanamycin (100 mg/liter), penicillin (100 mg/liter), and cycloheximide (100 mg/liter) were added to the medium, and N₂ was in the gas phase. TTY medium (27) with N₂ in the gas phase was used to determine methane production by mini-methane producers in ground stone. Inhibitors of methanogenic archaea were not added to the TTY medium. All samples were stored at 28°C in the dark. Methane production was monitored for 6 weeks. Autoclaved samples were used as controls.

RESULTS

Masonry, a habitat for methanotrophs.

Methanotrophs were present in 48 of 225 (almost 25%) stone samples from 19 buildings composed of the following types of stone: sandstone (13 samples), limestone (11 samples), marble (1 sample), lava (4 samples), granite (1 sample), brick (7 samples), plaster (2 samples), and mortar (9 samples). Methanotrophs were not found in the remaining samples of the types of stone listed above (75 sandstone, 39 limestone, 2 marble, 2 lava, 27 brick, 5 plaster, and 24 mortar samples), or in any of the 3 samples of terra-cotta. No relationship between the occurrence of methanotrophs and the type of stone material could be observed.

Cell numbers and activities of methanotrophs in stone material.

The cell numbers of methanotrophs in stone samples ranged between 5 and 86 CFU g of stone⁻¹. They were determined in stone samples in which the presence of methanotrophs had previously been shown (see above). The cell numbers might have been underestimated because the bacteria probably grow in the form of microcolonies which are associated with various groups of microorganisms in the form of a biofilm. Microcolonies can consist of 1 to 100 single cells and can be strongly attached to the stone material or exopolymeric substances (36). In this case the MPN technique counts a microcolony as a single cell.

The methane-oxidizing activities of methanotrophs in 12 selected stone samples were 11 to 42 pmol of CH₄ g of stone⁻¹ day⁻¹. High methane oxidation rates were generally correlated with high cell numbers of methanotrophs. No methane consumption was detected in the autoclaved controls.

Characterization of methanotrophs.

Twelve strains of methanotrophs were isolated from stone samples from historical buildings in Hanover (H strains), Halberstadt, Magdeburg, Potsdam, and Sicily (SZ strains). All strains were gram negative and possessed intracytoplasmic membranes arranged at the periphery of the cells. The isolates used the serine pathway for formaldehyde fixation and were able to produce sMMO under copper-limiting conditions. The sMMO activities ranged between 5 and 175 nmol of naphthol mg of protein⁻¹ min⁻¹. All isolates proved to be able to grow on 0.01% (vol/vol) methanol. Only the strains from Magdeburg, Sicily, and Hanover were able to grow on 1% methanol (vol/vol). None of the methanotrophs could grow at 45°C.

Due to their characteristics, the strains from Halberstadt, Magdeburg, and Potsdam belong to the genus Methylocystis. Only the isolate SZ 3/1 was able to grow heterotrophically and is therefore categorized as a facultative methylotrophic bacterium of the genus Methylobacterium. The strain H 6 belongs to the genus Methylosinus because of its ability to form exospores. All isolates were categorized as type II methanotrophs. The isolation method was not selective for type II, since Methylomonas methanica (type I) and Methylococcus capsulatus (type X) were able to grow under the applied isolation conditions.

Occurrence of methane-producing bacteria in masonry.

The presence of methane-producing bacteria on stone surfaces was not expected because of the direct contact with atmospheric oxygen. Nevertheless, methane production was detectable under nonoxic conditions. Table 1 shows the presence of methanotrophs and methane producers in 47 stone samples. About 45% of the samples showed no methane-oxidizing or -producing activity. Methanotrophs were found in 11 samples. Methane was produced in four samples with antibiotics added to the medium (32 to 215 nmol of CH₄ g of stone⁻¹ day⁻¹, for an average of 142 ± 63 nmol of CH₄ g of stone⁻¹ day⁻¹), indicating that the methane arose from methanogenic archaea. Small amounts of methane (0.07 to 1.6 nmol of CH₄ g of stone⁻¹ day⁻¹, for an average of 0.51 ± 0.3 nmol of CH₄ g of stone⁻¹ day⁻¹) were produced in 23 of the stone samples investigated in TTY medium without antibiotics, indicating the presence of heterotrophic mini-methane producers. Autoclaved controls did not produce methane under these conditions.

TABLE 1.

Occurrence of methanotrophs, mini-methane producers, and methanogenic archaea in 47 stone samples

Type of organism	No. of samples
None (no activity)	21
Methanotrophs	11
Methanogenic archaea	4
Mini-methane producers	23
Methanotrophs only	1
Methanotrophs and methanogenic archaea	4
Methanotrophs and mini-methane producers	10

A correlation between the presence of methanotrophs and methane producers was distinct. With only one exception, methanotrophs were found in samples which showed the presence of methane producers. This finding indicates that methanotrophs depend on biogenic methane production in or on stone surfaces.

DISCUSSION

Stone surfaces will be colonized by microorganisms if enough humidity and nutrients are available (4). The composition of colonies depends, in addition to other ecological factors, on the kind of stone material and nutrients available. Microorganisms occur on stone surfaces in the form of a biofilm which consists of different groups of organisms such as fungi, phototrophic and heterotrophic bacteria, nitrifiers, etc., and their exopolymeric substances (23). Biofilms have an influence on the water content of stone and enhance the adhesion of nutrients (23). The interaction of different groups of microorganisms present in stone has not been well examined. However, fungi are known to produce organic acids, like formate and acetate, which support growth of methanotrophic bacteria on stone surfaces (4, 35). Methanotrophs depend on C₁ compounds for growth, which are present in very low concentrations in rain or in the atmosphere (8). Atmospheric methane alone, normally present at a concentration of 1.7 ppm (vol/vol), cannot support growth of methanotrophs (6). However, locally increased concentrations of C₁ compounds in stones, caused by anthropogenic methane and methanol emissions or by endolithic methane producers, could be sufficient for growth of methanotrophs in masonry.

Methanotrophs were found in 48 of 225 stone samples. The absence of methanotrophs in some stone materials was probably due to the low sample number of these materials. The average cell number of methanotrophs in stone samples of 20 CFU g of stone⁻¹ was very low. In comparison, the cell numbers of oxic soils range between 10⁴ and 10⁷ CFU g of soil⁻¹ (2). This is probably because methanotrophs in soil are supplied with high methane concentrations from nonoxic parts of the soil. Corresponding to the low cell numbers of methanotrophs in stone, the methane-oxidizing activities also showed low values, 11 to 42 pmol of CH₄ g stone⁻¹ day⁻¹. However, if the activities of methanotrophs in stone samples are compared on the basis of cell numbers, they were in the same range as those in soils (2, 19).

For the first time we have demonstrated the presence of methanogenic archaea in stone. The methane production rates of the four positive samples were in the range of rates for methanogenic archaea in soil (10, 11). Organic acids like acetate or formate could be the electron donors for methanogens in anaerobic microsites of stones. Additionally, mini-methane producers were present in almost half of the 47 stone samples examined. This is consistent with the isolation by Schmidt of a mini-methane-producing Clostridium strain from stone material (29). Methane production in anaerobic microsites of aerobic biotopes, such as lakes and oxic soils, is well described (21, 24, 32). A high rate of oxygen consumption by aerobic microorganisms and a high water content should favor the formation of anaerobic niches in stone surfaces.

Our findings indicate that methanotrophs and methane producers belong to the autochthonic flora of building stones. It is likely that methanotrophs can use local methane produced by methane-producing bacteria as a carbon and energy source.

Exclusively type II methanotrophs of the genera Methylosinus and Methylocystis and a facultative type II methylotroph of the genus Methylobacterium were isolated from stone samples. Type I and type X methanotrophs were not isolated. Graham et al. (15) compared factors which affected the competition between type II and type I methanotrophs isolated from aquatic and terrestrial biotopes. Type II bacteria seem to be present at oligotrophic sites under nitrogen-limited conditions. In contrast, type I methanotrophs seem to dominate in methane-enriched locations. The isolated methanotrophs were able to produce sMMO under copper-limiting conditions. A high affinity for methane (K_m value of 1 μM CH₄) was determined for sMMO of Methylosinus trichosporium OB3b (18), while the K_m value of particulate MMO of the same strain was 66 μM CH₄ (30). This suggests that sMMO could be prerequisite for methane oxidation in oligotrophic locations like stone surfaces. Additionally, the isolates were able to utilize methanol at a concentration of 0.01% (vol/vol). It is not known whether methanol is oxidized by methanotrophs in masonry, because of the high numbers of facultative methylotrophic bacteria which compete for methanol at this location (3).