The DPRB Dechloromonas agitata CKB was grown at 30°C under N₂ on 20 mM sodium acetate and 10 mM NaClO₄ using phosphate buffer media (PBM) consisting of the following salts in solution (g/liter): Na₂HPO₄ (0.971), NaH₂PO₄ (0.379), NH₄Cl (0.25) plus 10 ml/l vitamins and 10 ml/l mineral stock solution (Sun et al., 2009). Methylococcus capsulatus Bath, Methylosinus trichosporium OB3b, and Methylomicrobium album BG8, were grown and maintained at 30°C on air + 30 kPa CH₄ using nitrate mineral salts media (NMS; Whittenbury et al., 1970). Cultures (1 l) for washed cell suspensions were harvested during late exponential phase, centrifuged (7000 × g), and washed twice with medium lacking substrates and vitamins. Final suspension volumes ranged from 5 to 150 ml. Cell concentrations at the start of incubations ranged from 1.8 × 10⁸ cells ml⁻¹ to 6.9 × 10⁸ cells ml⁻¹.

Soil from the seasonally exposed shoreline of Searsville Lake previously shown to harbor methanotrophic activity (Oremland and Culbertson, 1992) was air dried for two days at room temperature before sieving (<1 mm) to assure uniformity of soil particle size. Dried soil was stored for several weeks in stoppered 1 l glass flasks with air headspace and periodically augmented with 0.2 kPa CH₄ after consumption had removed all of the previously added CH₄ (4–6 days). Soil with thusly enhanced methanotrophic activity was used to determine CH₄ uptake in studies with added ClO⁻₄ and ClO⁻₂ in the absence of O₂.

Sediment slurries were prepared by adding 100 ml SeFr2 freshwater media (flushed with 20 kPa CO₂/80 kPa N₂; Miller et al., 2013) to 10 g Searsville Lake soil in N₂ flushed serum bottles (160 ml). Slurry pH was adjusted to 7.1 using 1 ml of 1 M NaHCO₃. Slurries were incubated under N₂ headspace following periodic amendments with 1 to 2 mmoles acetate and 0.5 to 1 mmole ClO⁻₄. Slurries were periodically sampled by syringe using 22 g needles and filtered through a 0.2 um Spin-X centrifuge tube. Acetate and ClO⁻₄ amendments were made after both were depleted (usually several days to weeks) during which time copious quantities of CH₄ were produced. Slurries with enhanced perchlorate reducing activity were used to determine methane uptake activity in the presence or absence of added O₂.

Aliquots of washed cell suspension of D. agitata CKB were distributed into stoppered and N₂ flushed 25 ml Balch tubes containing 10 ml PBM amended with 5 mM acetate and 10 mM NaClO₄, NaClO₃, or NaClO₂. Initial cell densities were 1.8 × 10⁸ cells per ml. The headspace was sampled over 7 days by syringe for CO₂ and O₂. Aqueous samples (0.3 ml) were collected by syringe for analysis of dissolved acetate and anions (Cl⁻, ClO⁻₄, ClO⁻₃, and ClO⁻₂). A short-term (10 min) experiment was conducted to follow ClO⁻₂ disproportionation. In this study, triplicate samples were sacrificed at pre-determined times. Activity was stopped by addition of 0.1 ml 4N NaOH before measurement of headspace O₂. Aqueous samples were subsequently collected for analysis of dissolved anions (Cl⁻ and ClO⁻₂).

Mixtures (10 ml) of M. capsulatus Bath and D. agitata CKB were prepared by adding washed cell suspensions of the cultures together in N₂ flushed Balch tubes (25 ml) sealed with butyl rubber stoppers. Methane (0.2 kPa) was introduced by syringe to all tubes and NaClO₂ (5 mM) was added to 3 tubes at the start of the incubation which was conducted at 37°C. Headspace CH₄ was monitored over 1 day. Single tubes were prepared without addition of ClO⁻₂ or without one of the cultures (i.e., no D. agitata CKB or no M. capsulatus Bath) to act as negative controls. A tube containing only M. capsulatus Bath under an air headspace acted as a positive control.

Additional microcosms were prepared in serum bottles (37 or 67 ml) using washed cell suspensions of D. agitata CKB and M. trichosporium OB3b or M. album BG8. Inocula were either combined in bottles (5 ml each) in one aqueous phase or kept separate by placing methanotrophs (1 ml) inside an open-topped glass tube contained within the bottles before flushing with N₂ and later adding D. agitata CKB (5 ml) by syringe to the bottom of the bottles. In this manner, the cultures were segregated but shared a common headspace. Methane (0.2 kPa) was introduced by syringe to all bottles and NaClO₄, NaClO₃, or NaClO₂ (5 or 10 mM) was added aseptically to start the incubations which were conducted at 28°C. Headspace CH₄ and aqueous anions were monitored over time. Controls were prepared without additions of ClO⁻₂.

Washed cell suspensions (5 ml each) of D. agitata CKB and M. trichosporium OB3b were added together to N₂ flushed serum bottles (13 ml). Radiolabeled ¹⁴CH₄ (5 μCi; specific activity = 21 μCi/μmole) was added along with 1 kPa CH₄ to the headspace of each bottle. Perchlorate (5 mM) was added to triplicate bottles and ClO⁻₂ (5 mM) was added to a single bottle by syringe to start the incubation which was conducted at 30°C. Gas samples for analysis of ¹⁴CH₄ and ¹⁴CO₂ were collected by syringe. At the end of the incubation, samples were acidified using 0.5 ml of 1.2N HCl to cause dissolved inorganic carbon (DIC = HCO⁻₃ + CO⁻²₃) to react to form CO₂ gas which was partitioned into the headspace. The headspace was again sampled by syringe. Control incubations consisted of single bottles of M. trichosporium OB3b alone under an air headspace and D. agitata CKB alone under N₂.

Slurry microcosms were prepared in N₂ flushed serum bottles (57 ml) containing 5 g of dried Searsville Lake soil with enhanced methanotrophic activity to which 10 ml Searsville Lake sediment slurry with enhanced perchlorate reducing activity (above) was added. Half the bottles were maintained under N₂ while half were flushed with air. Substrate (5 mM ClO⁻₄ or ClO⁻₂) was added by syringe followed by 0.5 ml CH₄ (1 kPa). Incubations were conducted at 22°C. Headspace and liquid samples were collected by syringe over 9 days.

Soil microcosms were prepared in serum bottles (67 ml) using washed cell suspensions of D. agitata CKB and dried Searsville Lake soil which was enhanced in methanotrophic activity (above). Soil (2 g) was placed inside open-topped glass tubes contained within the bottles prior to sealing and flushing with N₂. Subsequently, D. agitata CKB (10 ml) was added by syringe to the bottom of the bottles followed by aseptic addition of 5 mM acetate. The culture and the soil were thus segregated under a common headspace. Methane (0.1 kPa) was introduced by syringe to all bottles and 10 mM NaClO4, NaClO₃, or NaClO⁻₂ was added to D. agitata CKB to start the incubations. Incubations were conducted at 22°C. Headspace CH₄ and CO₂ and aqueous acetate and anions were monitored over 7 days.

Headspace O₂ was determined by ECD-GC using a molecular sieve 5A column (3.2 mm O.D. × 2.4 m) operated at 75°C using hydrocarbon-free UHP N₂ carrier. Background O₂ was minimized by flushing syringes and needles with O₂-free N₂ prior to sampling. The detection limit was 0.05 mmol O₂/L. Headspace CH₄ and CO₂ were determined by FID- and TCD-GC, respectively (Miller et al., 2013). Cell densities were determined by direct cell counting of liquid samples using acridine orange epi-fluorescence microscopy (Hobbie et al., 1977). Additional aqueous samples, including slurries, were filtered using Spin-X centrifuge filter tubes (0.2 μm; Corning Inc., Corning, NY) before determination of dissolved acetate by HPLC (Hoeft et al., 2004) or anions by IC (Miller et al., 2003). Dissolved ClO⁻₄ was analyzed separately by suppressed conductivity IC using a Dionex ISC 1100 containing an AS16 analytical column (4 × 250 mm) and an AG16 guard column (4 × 50 mm) with 0.035 M NaOH eluent. Measurements of headspace ¹⁴CH₄ and ¹⁴CO₂ were made by gas proportional counting (Culbertson et al., 1981) following TCD-GC analysis of CH₄ and CO₂ with separation on a Hayesep D column (100/120; 3.2 mm O.D. × 4.8 m) using UHP He carrier.

The total amount of gas in each bottle or tube was calculated from the headspace concentration using Henry's Law and the volumes of gas and liquid present. The dimensionless Henry's Law constants (K_H = C_G/C_L) used were 31.43 for O₂, 29.46 for CH₄ and 1.20 for CO₂ and were not corrected for ionic strength.

Dissimilatory (per)chlorate reduction by D. agitata CKB resulted in conversion of 85–100 μmoles added ClO⁻₄ or ClO⁻₃ to Cl⁻ in the presence of 50 μmoles added acetate (Figures 1A,B). Much less ClO⁻₄ or ClO⁻₃ (<15 μmoles) was consumed without added acetate and a corresponding lesser amount of Cl⁻ was produced. These observations suggest endogenous metabolism of intrinsic electron donors such as glycogen or polyhydroxybutyrate (PHB). No activity was observed in killed controls or in incubations with media and chloroxyanions alone (data not shown). Biological reduction of ClO⁻₄ or ClO⁻₃ and consumption of acetate occurred over approximately 2 days. Chlorite dismutation was much more rapid. More than half of the 100 μmoles ClO⁻₂ added was consumed and converted to Cl⁻ before the initial sampling at T = 2 min (Figure 1C). An additional 20 μmoles ClO⁻₂ were consumed over 7 days, however more than 20 μmoles ClO⁻₂ remained unreacted at the end.

Carbon dioxide (CO₂) was the dominant gaseous product of dissimilatory reduction of ClO⁻₄ or ClO⁻₃ in the presence of acetate (Figures 2A,B). Slightly more CO₂ was produced than could be accounted for by the added acetate. Little CO₂ was produced without added acetate. Small amounts of O₂ (up to 15 μmoles) were produced during incubations with ClO⁻₄ or ClO⁻₃. In contrast, ClO⁻₂ dismutation resulted in substantial and rapid O₂ production (Figure 2C) corresponding to release of >35% of the added ClO⁻₂ within the first day. As expected, there was no effect of added acetate on disproportionation of ClO⁻₂; however details of the early evolution of O₂ were obscured by the coarse sampling schedule.

The pattern of early O₂ production during ClO⁻₂ dismutation was made clear in a subsequent short-term (10 min) experiment where 56 μmoles of both O₂ and Cl⁻ were produced during the consumption of 56 μmoles of ClO⁻₂ (Figures 3A,B). A minor amount of CO₂ (<1 μmole) was produced (Figure 3C). Nearly half (40 μmoles) of the added ClO⁻₂ remained unreacted at the end of the experiment.

Methane was oxidized by methanotrophic bacteria M. capsulatus Bath (Figure 4A) and M. album BG8 (Figure 4B) during the reaction of D. agitata CKB with ClO⁻₂. No removal of CH₄ was observed in mixed cell suspensions amended with ClO⁻₄ or ClO⁻₃ (i.e., no added ClO⁻₂). Methane consumption occurred while the methanotrophs were in direct contact with up to 10 mM ClO⁻₂. Methane removal during incubations of co-cultures of D. agitata CKB with M. capsulatus Bath at 37°C occurred within 1 day while removal of CH₄ by M. album BG8 at 28°C occurred over 4 days. Similar rates of CH₄ consumption were observed for these mixed cultures whether they were segregated or co-mingled (Figure 4B) indicating that direct contact of cells was not required for methane consumption to occur.

Strain M. trichosporium OB3b co-cultured with D. agitata CKB containing 5 mM acetate oxidized ¹⁴CH₄ directly to ¹⁴CO₂ (Figure 5). The rate of ¹⁴CH₄ loss under anaerobic conditions with 5 mM added ClO⁻₂ was similar to that under aerobic conditions. No loss of ¹⁴CH₄ occurred in mixed cell suspensions amended with 5 mM ClO⁻₄ in lieu of ClO⁻₂ or in controls without methanotrophs. During the incubation, the product of methanotrophy (¹⁴CO₂) was distributed about equally between liquid and gas phases. Roughly 20% of the added ¹⁴CH₄ appeared as ¹⁴CO₂ in the headspace after 20 h (0.8 days). Most of the ¹⁴CH₄ added was recovered as ¹⁴CO₂ (60–90% recovery after acidification).

Searsville Lake sediment slurries removed repeated pulses of added ClO⁻₄ during anaerobic incubations using freshwater media with added acetate. Over a period of 1 month, 4 additions of 10 mM ClO⁻₄ were removed in bottles with commensurate consumption of 4 additions of 5 mM added acetate (data not shown). These slurries, thus enhanced in ClO⁻₄ reducing capacity, were used in separate incubations with added CH₄ (Figure 6). Under aerobic conditions, incubations with or without additions of ClO⁻₄ or ClO⁻₂ (5 mM) completely consumed 30 μmoles CH₄ within 5 days. However, no oxidation of CH₄ occurred during anaerobic incubations of Searsville Lake sediment slurries either with or without additions of ClO⁻₄ or ClO⁻₂.

Methane oxidation was observed when Searsville Lake soil (previously enhanced in methanotrophic activity) was segregated from liquid cultures of D. agitata CKB. Methane was completely consumed over the next 5 days by soil methanotrophs during the reaction of DPRB with 10 mM ClO⁻₂ (Figure 7). No CH₄ loss was observed when DPRB were provided with either 10 mM ClO⁻₄ or ClO⁻₃.

The Guest Associate Editor Hans Carlson declares that, despite having collaborated with author John Coates, the review process was handled objectively. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.