Characterization of a halotolerant acetoclastic methanogen highly enriched from marine sediment and its application in removal of acetate

Abstract

A marine sediment collected from Hiroshima Bay was cultured in artificial seawater, containing 0.51 M NaCl and 60 mM acetate and was found to exhibit active methane production at 37°C. Following four successive serial dilutions of cultures in medium containing 0.51 M NaCl, 60 mM acetate, and antibiotics, the well-acclimated methanogen was found to exhibit growth over a range of NaCl concentration (between 0 M and 2.06 M). The specific growth rates of the highly enriched methanogen, termed strain HA, in the absence of NaCl and in the presence of 1.54 M NaCl were estimated to be 0.037 h(-1) and 0.027 h(-1), respectively. The pH and temperature for optimum growth were determined to be 7.0-8.8 and 37°C, respectively. Although cells that had morphology similar to Methanosaeta sp. became dominant in the culture, methane production was still detected in the medium containing 0.51 M NaCl and other substrates such as methanol, formate, and methylamine, indicating contamination with other methanogens. The phylogenetic tree based on 16S rRNA gene sequences revealed that the strain HA was closely related to Methanosaeta harundinacea 6Ac and 8Ac(T), with sequence similarity of 98% and 97%, respectively. The continuous removal of acetate with upflow anaerobic filter reactor for industrial use of strain HA determined a methane production rate of 70 mM/d under condition of 0.51 M NaCl and successful methane production even under 1.54 M NaCl.

The Historical Development of Cultivation Techniques for Methanogens and Other Strict Anaerobes and Their Application in Modern Microbiology

“…The technique is based on serial dilutions in liquid media, ensuring the loss of most cells until the desired cells are the only present. This method is widely used, although it requires number of cultivation vessels to perform the method [71,117,118]. To remove contaminating microorganisms from an enrichment culture containing methanogens, the addition of appropriate antibiotics, such as ampicillin, vancomycin, clindamycin or kanamycin, could be used to simplify the process of purification [54,66,119].…”

Bioelectrochemical Stimulation of Electromethanogenesis at a Seawater-Based Subsurface Aquifer in a Natural Gas Field

“…In addition, the associated abrupt drops of both anodic and cathodic CEs were only shown after the opencircuit perturbation (Table 1). The SEM images of both anodic and cathodic biofilms revealed cylindrical microbes, similar in morphology to the marine Methanosaeta strain (Kita et al, 2016), only in reactor B (Supplementary Figure S8), and the cylindrical microbes did not adhere to the electrode surface as well as EET-active biofilms (Figure 10). These results imply that OTU BReur6 was not involved with EET on the electrodes (Figure 1A).…”

High-rate Fermentation of Acetate to Methane under Saline Condition by Aceticlastic Methanogens Immobilized in Marine Sediment

“…We previously enriched and identified aceticlastic halotolerant Methanosaeta sp. strain HA from the same marine sediment as that used in this study 20) . The morphology of this strain was quite similar to the Methanosaeta-like microorganisms observed by SEM in this study; however, the growth rate was lower than that of acclimated methanogens.…”

“…We recently reported that the aceticlastic methanogen Methanosaeta sp. starin HA, highly enriched from marine sediment collected from Hiroshima Bay, exhibited growth at 2.06 M (corresponding to 12 %) NaCl 20) . Therefore, we investigated continuous methane production from acetate by enriched Methanosaeta sp.…”

“…Therefore, we investigated continuous methane production from acetate by enriched Methanosaeta sp. strain HA in a glass column reactor packed with a polypropylene-ceramic mixture as support material for fixation of Methanosaeta 20) . In this set-up, methane production rate under 3 % NaCl was 70 mM d -1 .…”