Gelling and reducing agents are potential carbon and energy sources in culturing of anaerobic microorganisms.

Abstract

The majority of microorganisms in the environment have yet to be isolated in pure cultures, and the reasons behind this phenomenon remain elusive. In this study, we investigated the possibility of the commonly used gelling agent including agar and gellan gum as a source of carbon and energy in anaerobic roll-tube cultivation from one mangrove sediment sample and two high-temperature oilfield samples. Based on growth tests and genomic evidence, anaerobic gellan degraders were retrieved from genera of Clostridium, Lacrimispora, and lineages from the rarely cultivated phylum Atribacterota. Anaerobic agarolytic microorganisms were found to be members of Bacillus and Clostridium. We also proved the role of carbon and energy sources of L-cysteine, a routine agent used to make culture media anoxic/anaerobic in both enrichment cultures and isolated strains representing Acetomicrobium, Thermodesulfovibrio, Lacrimispora, Clostridium, Bacillus, Coprothermobacter, Citrobacter, and Enterobacter. Furthermore, the isolates and enriched microbial communities utilizing L-cysteine under anaerobic conditions were mainly through L-cysteine desulfuration to pyruvate, ammonia, and sulfide. This study demonstrates that the widely used gelling and reducing agents in the basal medium can serve as carbon and energy sources for anaerobic microorganisms and thus may bias the enrichment and isolation.

Importance: Most microbial species inhabiting natural environments have not been isolated in pure cultures using conventional media and laboratory conditions, but the reason behind this is unclear. Here, we provided a new explanation for the phenomenon, in that both the gelling agents, like agar and gellan gum, and reducing agent L-cysteine-HCl in the media provide extra carbon and energy sources to microorganisms and therefore decrease the chance in isolation specifically for the supplemented substrate which is supposed to be the sole source of carbon and energy. This result demonstrated that further improvement in the effectiveness of isolation of targeted microorganisms will be facilitated by subtracting the overlooked organic ingredients in the medium and more innovations.