Miniaturized extinction culturing is the preferred strategy for rapid isolation of fast-growing methane-oxidizing bacteria

Abstract

Methane-oxidizing bacteria (MOB) have a large potential as a microbial sink for the greenhouse gas methane as well as for biotechnological purposes. However, their application in biotechnology has so far been hampered, in part due to the relative slow growth rate of the available strains. To enable the availability of novel strains, this study compares the isolation of MOB by conventional dilution plating with miniaturized extinction culturing, both performed after an initial enrichment step. The extinction approach rendered 22 MOB isolates from four environmental samples, while no MOB could be isolated by plating. In most cases, extinction culturing immediately yielded MOB monocultures making laborious purification redundant. Both type I (Methylomonas spp.) and type II (Methylosinus sp.) MOB were isolated. The isolated methanotrophic diversity represented at least 11 different strains and several novel species based on 16S rRNA gene sequence dissimilarity. These strains possessed the particulate (100%) and soluble (64%) methane monooxygenase gene. Also, 73% of the strains could be linked to a highly active fast-growing mixed MOB community. In conclusion, miniaturized extinction culturing was more efficient in rapidly isolating numerous MOB requiring little effort and fewer materials, compared with the more widely applied plating procedure. This miniaturized approach allowed straightforward isolation and could be very useful for subsequent screening of desired characteristics, in view of their future biotechnological potential.

Figure 1

GTG₅ rep‐PCR fingerprinting using Pearson product moment correlation coefficient and UPGMA. MOB isolates were divided into 11 groups: 5 isolates showing unique profiles (B1, D1, E1, H1 and I2) and 6 groups of isolates (1–6) showing identical profiles. F1 and F2 isolates belonged to the genus Methylosinus, all the other isolates were identified as members of Methylomonas (Table 1). A representative of each cluster was selected randomly (indicated in bold) for further characterization.

Figure 2

DGGE analysis, based on the pmoA primer set A189fGC/mb661r for type I and type II MOB respectively. The community in the active enrichment from one sequence batch reactor is shown after the first cycle for each of the four samples, i.e. a sample from the wastewater treatment plant (WWTP), a slurry pit, biofilter material and a wetland. The 11 representative strains selected based on rep‐PCR fingerprinting (Fig. 1) are shown in relation to their corresponding active enrichment. Strains NCIMB11130^T (Methylomonas methanica, band indicated by arrow) and DSM17706^T (Methylosinus sporium) are shown as reference. Based on the band position of the strains, eight out of 11 representative strains could be linked to their active community, demonstrating the abundance of these strains in a mixed community selecting for fast‐growing methane‐oxidizing bacteria.

Figure 3

Flow chart of the followed methodology.